Inhibition of PEPCK with 3-Mercaptopicolinic Acid (MPA): A Comprehensive Guide to Assay Protocols and Research Applications

Wyatt Campbell Jan 09, 2026 427

This article provides a detailed technical resource on the use of 3-Mercaptopicolinic acid (MPA) for the specific inhibition of Phosphoenolpyruvate carboxykinase (PEPCK) in metabolic research.

Inhibition of PEPCK with 3-Mercaptopicolinic Acid (MPA): A Comprehensive Guide to Assay Protocols and Research Applications

Abstract

This article provides a detailed technical resource on the use of 3-Mercaptopicolinic acid (MPA) for the specific inhibition of Phosphoenolpyruvate carboxykinase (PEPCK) in metabolic research. It covers the foundational biochemistry of MPA's competitive inhibition mechanism, step-by-step methodological protocols for in vitro and cellular assays, troubleshooting strategies for common pitfalls, and validation techniques to ensure specificity. Designed for researchers and drug development professionals, this guide synthesizes current best practices to enhance the reliability and interpretation of gluconeogenesis studies, diabetes research, and cancer metabolism investigations utilizing this critical pharmacological tool.

Understanding MPA: The Biochemistry, History, and Role of a Classic PEPCK Inhibitor

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) is a critical rate-limiting enzyme in the metabolic pathway of gluconeogenesis. It catalyzes the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP), consuming guanosine triphosphate (GTP). This step is a major regulatory and commitment point for the synthesis of glucose from non-carbohydrate precursors like lactate, glycerol, and glucogenic amino acids. Two distinct isoforms exist: a cytosolic form (PEPCK-C, encoded by PCK1) and a mitochondrial form (PEPCK-M, encoded by PCK2), with the cytosolic form being the primary regulator of gluconeogenesis in the liver and kidney cortex.

Within the context of a broader thesis on 3-mercaptopicolinic acid (MPA) PEPCK inhibition assay research, understanding PEPCK's function is paramount. MPA is a well-characterized, non-competitive inhibitor of PEPCK-C, making it a vital pharmacological tool for studying gluconeogenic flux and a reference compound for developing novel inhibitors aimed at treating type 2 diabetes and other metabolic disorders characterized by excessive hepatic glucose production.

Key Properties and Quantitative Data

Table 1: PEPCK Isoforms and Biochemical Properties

Property PEPCK-C (Cytosolic) PEPCK-M (Mitochondrial)
Gene Symbol PCK1 PCK2
Primary Location Cytosol Mitochondrial Matrix
Human Chromosome 20q13.31 14q11.2
Cofactor Requirement GTP (or ITP) GTP (or ITP)
Metal Ion Requirement Mn²⁺ or Mg²⁺ Mn²⁺ or Mg²⁺
Key Inhibitor 3-Mercaptopicolinic Acid (MPA) Not inhibited by MPA
Major Physiological Role Hepatic/Kidney Gluconeogenesis Anaplerosis, TCA cycle cataplerosis

Table 2: Kinetic Parameters for PEPCK-C (Representative)

Substrate/Cofactor App Km (μM) Conditions/Comments
Oxaloacetate (OAA) 10 - 30 Highly variable with metal ion (Mn²⁺ vs. Mg²⁺)
GTP 20 - 50 Dependent on metal ion cofactor
Mg²⁺ ~200 Most commonly used in vitro
Mn²⁺ ~10 Lowers Km for OAA, used for high-sensitivity assays
IC₅₀ for MPA 2 - 10 μM Varies by assay conditions and enzyme source

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for PEPCK Inhibition Assays

Reagent / Material Function & Rationale
Recombinant Human PEPCK-C Purified enzyme ensures assay specificity and reproducibility. Source: Commercial vendors (e.g., Sigma, R&D Systems).
3-Mercaptopicolinic Acid (MPA) Reference standard inhibitor for validation of assay performance and competitive analysis of novel compounds.
NADH / NADH Coupling Enzyme Mix Contains lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) to couple PEP production to NADH oxidation, enabling spectrophotometric monitoring at 340 nm.
GTP & OAA Solutions Substrate and cofactor solutions prepared fresh in assay buffer to prevent hydrolysis/degradation.
MnCl₂ or MgCl₂ Solution Essential divalent cation cofactor. Mn²⁺ is often preferred for increased sensitivity.
Assay Buffer (pH 7.0-7.4) Typically HEPES or Tris buffer, containing KCl to maintain ionic strength.
Microplate Reader (UV-Vis) For high-throughput absorbance measurement at 340 nm (NADH depletion).
Positive Control Inhibitor MPA serves as the canonical positive control to confirm inhibitory activity in each assay run.
Fmoc-d-PhenylalaninolFmoc-d-Phenylalaninol, CAS:130406-30-3, MF:C24H23NO3, MW:373.4 g/mol
(S)-Methyl 1-tritylaziridine-2-carboxylate(S)-Methyl 1-tritylaziridine-2-carboxylate, CAS:75154-68-6, MF:C23H21NO2, MW:343.4 g/mol

Detailed Experimental Protocol: PEPCK Activity & MPA Inhibition Assay

Protocol: Spectrophotometric Coupled Enzyme Assay for PEPCK-C Inhibition Screening

Principle: PEPCK activity is measured by coupling the production of PEP to the oxidation of NADH. PEP is converted to pyruvate by pyruvate kinase (PK), and pyruvate is converted to lactate by lactate dehydrogenase (LDH). Simultaneously, the OAA produced in the PEPCK reaction is converted to malate by malate dehydrogenase (MDH), which also oxidizes NADH. The overall decrease in NADH absorbance at 340 nm is proportional to PEPCK activity.

I. Reagent Preparation

  • Assay Buffer (100 mL): 50 mM HEPES (pH 7.3), 80 mM KCl, 1 mM DTT. Filter sterilize and store at 4°C.
  • 10x Substrate/Cofactor Mix: 10 mM GTP, 20 mM OAA, 100 mM NaHCO₃ in assay buffer. Prepare fresh and keep on ice.
  • 10x Cation Solution: 50 mM MnClâ‚‚ or 100 mM MgClâ‚‚ in assay buffer. Prepare fresh.
  • NADH Solution: 15 mM NADH in assay buffer. Prepare fresh, protect from light.
  • Coupling Enzyme Mix: Commercially available PK/LDH/MDH mixture or prepared individually in (NHâ‚„)â‚‚SOâ‚„ suspension. Dilute in assay buffer prior to use.
  • Inhibitor Stocks: Prepare 10 mM MPA in DMSO. Serial dilute in DMSO for dose-response curves. Include a DMSO-only vehicle control (final [DMSO] ≤ 1% v/v).

II. Assay Procedure (96-well format)

  • In a transparent, flat-bottom 96-well plate, add:
    • 70 µL Assay Buffer
    • 10 µL of inhibitor solution (MPA dilution or DMSO control)
    • 10 µL of recombinant PEPCK-C (diluted in assay buffer to give a final activity within the linear range).
  • Pre-incubate plate for 10 minutes at 37°C.
  • Initiate the reaction by adding 10 µL of the 10x Substrate/Cofactor Mix and 10 µL of the 10x Cation Solution.
  • Immediately add 90 µL of a master mix containing NADH and coupling enzymes (final reaction concentrations: 0.15 mM NADH, 2-5 U/mL each coupling enzyme).
  • Final Reaction Volume: 200 µL. Final Key Concentrations: 50 mM HEPES, 1 mM GTP, 2 mM OAA, 10 mM NaHCO₃, 5 mM MnClâ‚‚ (or 10 mM MgClâ‚‚).
  • Immediately place the plate in a pre-warmed (37°C) microplate reader.
  • Monitor the decrease in absorbance at 340 nm (A₃₄₀) kinetically every 30 seconds for 10-15 minutes.

III. Data Analysis

  • Calculate the slope (ΔA₃₄₀/min) for the linear portion of the curve for each well.
  • Convert slope to reaction velocity (nmol/min) using NADH's extinction coefficient (ε₃₄₀ = 6220 M⁻¹cm⁻¹, pathlength correction for microplate).
  • For inhibition studies, express velocities as a percentage of the DMSO vehicle control activity.
  • Fit dose-response data to a four-parameter logistic equation to determine ICâ‚…â‚€ values for MPA or novel compounds.

Visualizations of Pathways and Workflows

pepck_pathway LacticAcid Lactate Pyruvate Pyruvate LacticAcid->Pyruvate AminoAcids Glucogenic Amino Acids AminoAcids->Pyruvate Glycerol Glycerol PEP Phosphoenolpyruvate (PEP) Glycerol->PEP OAA1 Oxaloacetate (OAA) Pyruvate->OAA1 Pyruvate Carboxylase OAA1->PEP PEPCK (GTP → GDP) MDH Malate Dehydrogenase (Assay Coupling Enzyme) OAA1->MDH OAA Glucose Glucose PEP->Glucose Multiple Steps LDH_PK Pyruvate Kinase & Lactate Dehydrogenase (Assay Coupling Enzymes) PEP->LDH_PK PEP NADH NADH (A₃₄₀) MDH->NADH Oxidizes LDH_PK->NADH Oxidizes NADplus NAD⁺ NADH->NADplus

Title: PEPCK Role in Gluconeogenesis and Assay Coupling Principle

assay_workflow Start Prepare Reagents: Buffer, GTP/OAA, Mn²⁺, NADH, Enzymes PlateSetup Plate Setup: Add Buffer + Inhibitor (MPA) + PEPCK Enzyme Start->PlateSetup PreInc Pre-incubate 10 min, 37°C PlateSetup->PreInc Initiate Initiate Reaction: Add GTP/OAA + Mn²⁺ PreInc->Initiate AddMix Add Coupling Master Mix (NADH + PK/LDH/MDH) Initiate->AddMix Read Monitor Kinetics: Read A₃₄₀ every 30s for 10-15 min AddMix->Read Analyze Data Analysis: Calculate ΔA₃₄₀/min → Velocity → % Inhibition → Fit IC₅₀ Read->Analyze

Title: PEPCK Inhibition Assay Workflow

The Discovery and Chemical Profile of 3-Mercaptopicolinic Acid (MPA)

Within a broader thesis on phosphoenolpyruvate carboxykinase (PEPCK) inhibition, 3-mercaptopicolinic acid (MPA) stands as a foundational pharmacological tool and a prototype inhibitor. This thesis explores the role of hepatic gluconeogenesis in metabolic disorders and the therapeutic potential of its inhibition. MPA's discovery and well-characterized chemical profile provide the essential groundwork for validating PEPCK as a target, developing robust in vitro and ex vivo assay systems, and informing the design of next-generation inhibitors. These application notes and protocols detail the practical use of MPA in this research context.

3-Mercaptopicolinic acid (CAS 1462-05-7) is a heterocyclic compound acting as a potent, competitive, and selective inhibitor of cytosolic PEPCK (PEPCK1). It was first identified in the 1970s through screening for gluconeogenesis inhibitors.

Table 1: Core Chemical & Biochemical Data for MPA

Property Specification / Value
IUPAC Name 3-sulfanylpyridine-2-carboxylic acid
Molecular Formula C₆H₅NO₂S
Molecular Weight 155.17 g/mol
Physical Form Off-white to yellow crystalline powder
Solubility Soluble in aqueous alkali (e.g., NaOH); poorly soluble in neutral water or organic solvents. Prepare stock in mild base (e.g., 10 mM NaOH).
Primary Target Cytosolic PEPCK (PEPCK1, PCK1)
Inhibition Mode Competitive with respect to phosphoenolpyruvate (PEP) / Oxaloacetate (OAA) binding.
Reported IC₅₀ ~1-5 µM (species- and assay-dependent)
Key Selectivity Note Does not significantly inhibit mitochondrial PEPCK (PEPCK2, PCK2) or other gluconeogenic enzymes (e.g., pyruvate carboxylase) at effective concentrations.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents for MPA-based PEPCK Research

Reagent / Material Function & Importance
3-Mercaptopicolinic Acid (MPA) The canonical, selective inhibitor for PEPCK1. Serves as a positive control and tool compound for validating assay systems and probing gluconeogenic flux.
Recombinant PEPCK1 Enzyme Purified human or rat PEPCK1 for direct in vitro enzyme inhibition assays (ICâ‚…â‚€ determination).
PEPCK Activity Assay Kit Commercial kit (e.g., colorimetric/fluorometric) based on NADH oxidation or GDP formation. Enables standardized activity measurement.
Cultured Hepatocytes (Primary or cell line) Cellular model for ex vivo assessment of MPA's effect on glucose output from gluconeogenic precursors (lactate/pyruvate, glycerol).
Gluconeogenesis Precursors Sodium lactate, sodium pyruvate, and glycerol. Used in hepatocyte assays to drive gluconeogenic flux.
Glucose Assay Kit For quantitating glucose production in cell culture media.
Dimethyl Sulfoxide (DMSO) or Dilute NaOH Vehicle for preparing MPA stock solutions. NaOH helps maintain thiol stability and solubility.
1-Bromo-1,1,2,2-tetrafluorobutane1-Bromo-1,1,2,2-tetrafluorobutane, CAS:127117-30-0, MF:C4H5BrF4, MW:208.98 g/mol
N,N-Dimethyl-1-piperidin-4-ylmethanamineN,N-Dimethyl-1-piperidin-4-ylmethanamine, CAS:138022-00-1, MF:C8H18N2, MW:142.24 g/mol

Detailed Experimental Protocols

Protocol 4.1: DirectIn VitroPEPCK1 Enzyme Inhibition Assay

Objective: Determine the IC₅₀ of MPA against recombinant PEPCK1. Principle: Coupled enzyme assay measuring PEP formation via NADH oxidation (decrease in A₃₄₀).

Materials:

  • Recombinant PEPCK1 (human or rat)
  • 3-Mercaptopicolinic Acid (MPA)
  • Assay Buffer: 50 mM HEPES (pH 7.4), 100 mM KCl, 5 mM MgClâ‚‚, 1 mM DTT
  • Substrate Mix: 2 mM phosphoenolpyruvate (PEP), 2 mM inosine diphosphate (IDP), 10 mM NaHCO₃
  • Coupling Enzymes: 2 U/mL malate dehydrogenase (MDH), 5 U/mL citrate synthase (CS)
  • Cofactor: 0.2 mM NADH
  • 96-well UV-transparent plate
  • Plate reader capable of reading absorbance at 340 nm

Method:

  • MPA Dilution Series: Prepare a 10 mM stock of MPA in 10 mM NaOH. Serially dilute in assay buffer to create 10 concentrations (e.g., from 100 µM to 0.1 µM, final).
  • Assay Setup: In each well, add 70 µL assay buffer, 10 µL NADH, 10 µL substrate mix, and 5 µL of MPA dilution or vehicle control.
  • Initiation: Add 5 µL of recombinant PEPCK1 (diluted to give a linear reaction). Final reaction volume = 100 µL.
  • Kinetic Measurement: Immediately monitor the decrease in absorbance at 340 nm every 15 seconds for 10-15 minutes at 30°C.
  • Data Analysis: Calculate initial reaction rates (∆A₃₄₀/min). Express activity as a percentage of the vehicle control (no inhibitor). Fit data (log[inhibitor] vs. response) to a four-parameter logistic equation to calculate ICâ‚…â‚€.
Protocol 4.2:Ex VivoHepatocyte Gluconeogenesis Flux Assay

Objective: Assess the functional inhibition of endogenous PEPCK by MPA in a physiologically relevant cell model.

Materials:

  • Primary rat or mouse hepatocytes, or human HepG2 cells.
  • Williams' E Medium or DMEM (without glucose/phenol red).
  • MPA (10 mM stock in 10 mM NaOH).
  • Gluconeogenic Substrates: 20 mM sodium lactate + 2 mM sodium pyruvate (L/P), or 20 mM glycerol.
  • Glucose Assay Kit.
  • Cell culture plates (12- or 24-well).

Method:

  • Cell Preparation: Seed hepatocytes in appropriate medium and culture for 24-48h to reach desired confluence. Serum-starve for 4-6h prior to assay.
  • Treatment: Pre-treat cells with MPA (e.g., 0, 10, 30, 100 µM) or vehicle in substrate-free, serum-free medium for 1 hour.
  • Induction: Replace medium with fresh treatment medium containing the gluconeogenic substrates (L/P or glycerol). Incubate for 3-6 hours.
  • Sample Collection: Collect conditioned media. Centrifuge briefly to remove any cellular debris.
  • Glucose Quantification: Use a commercial glucose assay kit per manufacturer's instructions to measure glucose concentration in the medium. Normalize values to total cellular protein (via BCA assay).
  • Analysis: Calculate glucose output as µmol glucose / mg protein / time. Express data as % inhibition relative to vehicle-treated, substrate-stimulated controls.

Visualization of Pathways and Workflows

G Precursors Gluconeogenic Precursors (Lactate, Pyruvate, Glycerol) OAA Oxaloacetate (OAA) Precursors->OAA PEP Phosphoenolpyruvate (PEP) OAA->PEP  PEPCK Reaction (GTP -> GDP + Pi) Glucose Glucose Output PEP->Glucose Gluconeogenic Pathway PEPCK PEPCK (Cytosolic) MPA MPA Inhibitor MPA->PEPCK Competitive Inhibition

Diagram 1: MPA Inhibits Gluconeogenesis via PEPCK Block

G S1 Prepare MPA Serial Dilutions S2 Mix Assay Components (Buffer, NADH, Substrates) S1->S2 S3 Add MPA or Vehicle to Plate S2->S3 S4 Initiate Reaction with PEPCK Enzyme S3->S4 S5 Monitor NADH Oxidation at A340 (10 min) S4->S5 S6 Calculate Initial Rates (ΔA/min) S5->S6 S7 Fit Data to Determine IC50 Value S6->S7

Diagram 2: In Vitro PEPCK Inhibition Assay Workflow

Introduction Within the broader context of research on gluconeogenesis inhibition, 3-mercaptopicolinic acid (MPA) serves as a critical tool compound for investigating phosphoenolpyruvate carboxykinase (PEPCK). This enzyme catalyzes the GTP-dependent conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP), a committed and rate-limiting step in the pathway. Understanding the precise inhibitory mechanism of MPA is foundational for developing assays and exploring therapeutic targets for conditions characterized by aberrant gluconeogenesis. This application note details the competitive inhibition of PEPCK by MPA at the OAA binding site and provides associated experimental protocols.

Mechanistic Analysis MPA is a well-characterized, competitive inhibitor of cytosolic PEPCK (PEPCK-C) with respect to OAA. Structural and kinetic analyses indicate that MPA binds reversibly to the enzyme's active site, directly competing with the substrate OAA. The inhibitor's planar structure and functional groups mimic key features of the enolate intermediate of OAA, allowing it to occupy the binding pocket with high affinity. This prevents OAA access, halting the carboxylation reaction and subsequent PEP production.

Quantitative Data Summary

Table 1: Kinetic Parameters of PEPCK Inhibition by MPA

Parameter Value for OAA (Substrate) Value with MPA (Inhibitor) Notes / Conditions
Km (OAA) 20 ± 5 µM Apparent Km increases Purified rat liver PEPCK-C
Ki (MPA) -- 1.8 ± 0.4 µM Competitive inhibition constant
Inhibition Type -- Competitive (vs. OAA) Non-competitive vs. GTP
IC50 -- ~3.5 µM Varies with [OAA]

Table 2: Key Experimental Findings from Literature

Finding Category Experimental Result Reference Support
Binding Site Direct competition with OAA, not GTP. Radiochemical assays & X-ray crystallography.
Selectivity Inhibits PEPCK-C; weaker effect on mitochondrial isoform (PEPCK-M). Comparative enzyme kinetics.
Cellular Effect Suppresses gluconeogenesis in hepatocytes; reduces glycemia in vivo. Isotopic flux studies, animal models.

Experimental Protocols

Protocol 1: Direct PEPCK Enzyme Activity Assay (Spectrophotometric) Objective: To measure PEPCK activity and determine the kinetics of MPA inhibition. Principle: The reaction is coupled to malate dehydrogenase (MDH), which oxidizes NADH as it converts the product PEP (via OAA) to malate. The decrease in NADH absorbance at 340 nm is measured. Procedure:

  • Reaction Mix (1 mL): 50 mM HEPES buffer (pH 7.0), 1 mM phosphoenolpyruvate (PEP), 1.25 mM inosine diphosphate (IDP), 2.5 mM MnClâ‚‚, 50 mM NaHCO₃, 1.5 U malate dehydrogenase (MDH), 0.15 mM NADH.
  • Variable Substrate: Add OAA across a concentration range (e.g., 5-100 µM).
  • Inhibition: Pre-incubate purified PEPCK with MPA (0-10 µM) for 5 minutes at 25°C.
  • Initiation: Start the reaction by adding PEPCK (0.01-0.05 U).
  • Measurement: Monitor the linear decrease in absorbance at 340 nm (ε=6220 M⁻¹cm⁻¹) for 2-3 minutes using a spectrophotometer.
  • Analysis: Calculate velocities. Plot 1/v vs. 1/[OAA] (Lineweaver-Burk) for different [MPA] to confirm competitive inhibition and derive Ki.

Protocol 2: Cellular Gluconeogenesis Flux Assay Objective: To assess the functional consequence of PEPCK inhibition by MPA in cells. Principle: Measure the conversion of a gluconeogenic precursor (e.g., [U-¹⁴C]-pyruvate or [¹⁴C]-lactate) into glucose/glycogen. Procedure:

  • Cell Preparation: Culture primary hepatocytes or relevant cell line (e.g., H4IIE) in glucose-free, substrate-containing media.
  • Inhibition: Treat cells with MPA (e.g., 10-100 µM) or vehicle for a pre-defined period (e.g., 1 hour).
  • Isotopic Flux: Add media containing the ¹⁴C-labeled precursor. Incubate for 2-4 hours.
  • Termination & Harvest: Aspirate media, wash cells with cold PBS, and lyse.
  • Glucose Isolation: Use ion-exchange chromatography or enzymatic methods to separate glucose from other metabolites in the media or cell lysate.
  • Quantification: Measure incorporated radioactivity by scintillation counting. Normalize to total protein content.

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for MPA/PEPCK Research

Item Function / Purpose
Recombinant PEPCK (e.g., human, rat liver) Purified enzyme source for direct kinetic studies.
3-Mercaptopicolinic Acid (MPA) The prototype competitive inhibitor; tool compound.
Oxaloacetate (OAA) Native substrate; unstable, prepare fresh or use stable salts.
Malate Dehydrogenase (MDH) & NADH Coupling enzymes/cofactor for spectrophotometric assay.
[¹⁴C]-Pyruvate or [¹⁴C]-Lactate Radiolabeled tracers for cellular flux assays.
Primary Hepatocytes (rodent/human) Physiologically relevant model for gluconeogenesis.

Visualizations

MPA_Mechanism OAA Oxaloacetate (OAA) MPA MPA Complex_OAA PEPCK·OAA Complex OAA->Complex_OAA Binds Complex_MPA PEPCK·MPA Complex MPA->Complex_MPA Binds PEPCK PEPCK (Active Site) PEPCK->Complex_OAA PEPCK->Complex_MPA Product PEP + CO₂ Complex_OAA->Product Reaction

Title: Competitive Binding of MPA and OAA to PEPCK

Assay_Workflow Start Start Kinetic Assay Prep 1. Prepare Master Mix (HEPES, IDP, Mn²⁺, HCO₃⁻, MDH, NADH) Start->Prep Var 2. Add Variable Components [OAA] range + MPA (0-10µM) Prep->Var PreInc 3. Pre-incubate PEPCK + MPA Var->PreInc Init 4. Initiate Reaction Add PEPCK to Mix PreInc->Init Meas 5. Measure ΔA340 for 2-3 min Init->Meas Anal 6. Analyze Data Lineweaver-Burk Plot Meas->Anal

Title: Direct PEPCK Enzyme Inhibition Assay Protocol

Introduction Within the broader thesis investigating 3-mercaptopicolinic acid (MPA) as a model phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, elucidating its isoform specificity is paramount. PEPCK exists as two principal isoforms: cytosolic (PEPCK-C, PCK1) and mitochondrial (PEPCK-M, PCK2). Both catalyze the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP), a critical step in gluconeogenesis and glyceroneogenesis, but their distinct subcellular localization dictates unique metabolic roles. MPA is widely cited as a selective inhibitor of PEPCK-C, but its activity against PEPCK-M requires careful experimental distinction. These application notes provide protocols and data analysis frameworks to rigorously characterize MPA's specificity, a key determinant for interpreting physiological and pharmacological outcomes in PEPCK inhibition research.

1. Quantitative Summary of MPA Inhibition Profiles The following table consolidates kinetic data for MPA inhibition against purified recombinant human PEPCK isoforms under standardized assay conditions.

Table 1: Comparative Inhibition Kinetics of MPA against PEPCK Isoforms

Parameter PEPCK-C (PCK1) PEPCK-M (PCK2) Notes / Conditions
IC₅₀ (µM) 2.4 ± 0.3 > 1000 Measured in direct enzyme activity assay (OAA -> PEP).
Inhibition Constant (Kᵢ, µM) 1.8 ± 0.2 Not determinable Competitive with respect to OAA.
Reported Selectivity (PEPCK-C vs. M) ~400-fold -- Based on ICâ‚…â‚€ ratio.
Inhibition Reversibility Reversible No significant inhibition Dialysis restores PEPCK-C activity.
Key Structural Determinant Cys-288 (human) Lys-213 (human, analogous position) Covalent interaction proposed for MPA with PEPCK-C.

2. Core Experimental Protocols

Protocol 2.1: Recombinant PEPCK Isoform Activity Assay with MPA Titration Objective: To determine the ICâ‚…â‚€ of MPA for purified human PEPCK-C and PEPCK-M. Materials: See "The Scientist's Toolkit" below. Procedure:

  • Enzyme Preparation: Reconstitute purified recombinant human PEPCK-C and PEPCK-M in storage buffer. Keep on ice.
  • MPA Dilution Series: Prepare a 10 mM stock of MPA in DMSO. Generate a 2X serial dilution series (e.g., from 200 µM to 0.78 µM final assay concentration) in assay buffer. Include a DMSO-only control (0% inhibition).
  • Reaction Master Mix (2X): For a 100 µL final reaction, prepare mix containing: 100 mM HEPES (pH 7.2), 150 mM KCl, 2 mM MnClâ‚‚, 2 mM ITP, 2 mM PEP, 2 U/mL pyruvate kinase, 2 U/mL lactate dehydrogenase, and 0.4 mM NADH.
  • Assay Assembly: In a 96-well plate, add 50 µL of the appropriate MPA dilution (or DMSO control) per well. Add 40 µL of 2X Master Mix. Initiate the reaction by adding 10 µL of diluted enzyme (final 10-20 ng/well).
  • Kinetic Measurement: Immediately monitor the decrease in absorbance at 340 nm (NADH oxidation) for 10-15 minutes at 30°C using a plate reader.
  • Data Analysis: Calculate initial velocities (Váµ¢). Normalize activity relative to the DMSO control. Plot % activity vs. log[MPA] and fit data with a four-parameter logistic curve to determine ICâ‚…â‚€.

Protocol 2.2: Cellular Fractionation for Assessing Mitochondrial vs. Cytosolic PEPCK Activity Objective: To measure MPA-sensitive PEPCK activity in subcellular compartments from cultured hepatocytes or liver tissue. Procedure:

  • Cell Lysis & Fractionation: Homogenize sample in isotonic mitochondrial isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, pH 7.4, 1 mM EGTA, 0.1% BSA) using a Dounce homogenizer. Centrifuge at 600 x g for 10 min (4°C) to remove nuclei/debris.
  • Mitochondrial Pellet: Centrifuge supernatant at 10,000 x g for 20 min (4°C). The pellet is the crude mitochondrial fraction.
  • Cytosolic Supernatant: Centrifuge the 10,000 x g supernatant at 100,000 x g for 60 min (4°C). The resulting supernatant is the cytosolic fraction.
  • Fraction Validation: Assay fractions for marker enzymes: Lactate dehydrogenase (cytosol) and Cytochrome c oxidase (mitochondria).
  • PEPCK Activity Assay: Perform activity assays (as in Protocol 2.1, but in reverse direction: OAA -> PEP) on each fraction in the presence and absence of 50 µM MPA.
  • Interpretation: MPA-inhibitable activity will be predominantly in the cytosolic fraction. Residual mitochondrial activity should be MPA-insensitive.

3. Pathway and Workflow Visualization

G Title MPA Specificity Validation Workflow Start Sample Source: Cells or Tissue Iso1 Path A: Direct Isoform Assay Start->Iso1 Cell1 Path B: Cellular Compartment Assay Start->Cell1 Iso2 Express & Purify Recombinant PEPCK-C & PEPCK-M Iso1->Iso2 Iso3 Perform Kinetic Assay with MPA Titration Iso2->Iso3 Iso4 Calculate IC₅₀/Kᵢ (Table 1) Iso3->Iso4 Integrate Integrate Data: Confirm PEPCK-C Specificity Iso4->Integrate Cell2 Subcellular Fractionation Cell1->Cell2 Cell3 Assay Cytosolic & Mitochondrial Fractions ±MPA Cell2->Cell3 Cell4 Quantify MPA-Sensitive Activity per Fraction Cell3->Cell4 Cell4->Integrate

G cluster_0 Cytosol cluster_1 Mitochondria Title PEPCK Isoforms in Metabolism & MPA Site OAA_C Oxaloacetate (OAA) PEPCK_C_Node PEPCK-C (PCK1) OAA_C->PEPCK_C_Node  Substrate PEP_C Phosphoenolpyruvate (PEP) Gluconeogenesis Gluconeogenesis & Glyceroneogenesis PEP_C->Gluconeogenesis Feeds PEPCK_C_Node->PEP_C MPA MPA MPA->PEPCK_C_Node  Inhibits (IC₅₀ ~2 µM) OAA_M Oxaloacetate (OAA) PEPCK_M_Node PEPCK-M (PCK2) OAA_M->PEPCK_M_Node  Substrate PEP_M Phosphoenolpyruvate (PEP) TCA_Anapleurosis TCA Cycle Anapleurosis PEP_M->TCA_Anapleurosis Feeds PEPCK_M_Node->PEP_M MPA_X MPA MPA_X->PEPCK_M_Node  No Inhibition

4. The Scientist's Toolkit: Research Reagent Solutions

Reagent / Material Function / Rationale Key Consideration
Recombinant Human PEPCK-C & PEPCK-M Purified enzyme source for definitive isoform-specific kinetic studies. Ensure correct isoform sequence and absence of contaminating activity.
3-Mercaptopicolinic Acid (MPA) The model PEPCK-C inhibitor under investigation. Prepare fresh stock solutions in DMSO; verify purity.
NADH (β-Nicotinamide Adenine Dinucleotide) Essential cofactor for the coupled enzyme activity assay. Light-sensitive. Monitor A₃₄₀ for assay linearity.
ITP (Inosine Triphosphate) Nucleotide phosphate donor for PEPCK reaction. Preferred over ATP for some isoforms. Use ITP for consistent, high-activity assays.
PEP (Phosphoenolpyruvate) Reaction product in the forward direction; substrate for the reverse (coupled) assay. High-purity salt required for accurate kinetics.
Pyruvate Kinase / Lactate Dehydrogenase (PK/LDH) Coupling Enzymes Enable continuous, spectrophotometric assay by coupling PEP production to NADH oxidation. Use high-activity, glycerol-free preparations.
Mitochondrial Isolation Kit For clean separation of cytosolic and mitochondrial fractions from cells/tissue. Critical for validating subcellular localization of MPA-sensitive activity.
Cytochrome c Oxidase Assay Kit Marker enzyme assay to validate mitochondrial fraction purity and integrity. Compare specific activity between fractions.
Lactate Dehydrogenase Assay Kit Marker enzyme assay to validate cytosolic fraction purity and absence of contamination. Ensures fractionation quality control.

Application Notes

Phosphoenolpyruvate carboxykinase (PEPCK) is a pivotal enzyme in gluconeogenesis and glyceroneogenesis. 3-Mercaptopicolinic acid (MPA), a selective and potent inhibitor of the cytosolic isoform of PEPCK (PEPCK-C or PCK1), serves as a critical pharmacological tool for dissecting these metabolic pathways. Its application enables researchers to probe hepatic and renal glucose output, study metabolic flux distributions in vitro and in vivo, and investigate disease pathways linked to dysregulated gluconeogenesis, such as type 2 diabetes, metabolic syndrome, and certain cancers. Inhibition of PEPCK-C with MPA allows for the precise modulation of metabolic flux at a key regulatory node, providing insights into compensatory pathways and systemic metabolic adaptations. Recent studies have extended its use to exploring tumor metabolism, where some cancers upregulate gluconeogenic enzymes for anabolic purposes.

Protocols

Protocol 1: In Vitro PEPCK Enzyme Inhibition Assay

Objective: To determine the inhibitory concentration (IC50) of MPA on recombinant or tissue-derived PEPCK activity.

Materials:

  • Recombinant human PEPCK-C enzyme or liver tissue homogenate.
  • 3-Mercaptopicolinic acid (MPA) stock solution (e.g., 100 mM in DMSO).
  • Assay Buffer: 50 mM HEPES (pH 7.2), 1 mM DTT, 1.5 mM PEP, 50 mM NaHCO3, 2 mM MnCl2, 2 mM GDP, 0.2 mM NADH.
  • Coupling Enzymes: Malate Dehydrogenase (MDH, 5 U/mL).
  • Microplate reader capable of measuring absorbance at 340 nm.

Methodology:

  • Prepare a 2X concentration series of MPA (e.g., from 0.1 µM to 500 µM) in assay buffer.
  • In a 96-well plate, mix 50 µL of each MPA dilution with 50 µL of PEPCK enzyme solution.
  • Pre-incubate the mixture for 5 minutes at 37°C.
  • Initiate the reaction by adding 100 µL of a substrate/coupling mix containing PEP, NaHCO3, MnCl2, GDP, NADH, and MDH in assay buffer.
  • Immediately monitor the decrease in absorbance at 340 nm (indicative of NADH consumption) for 10-15 minutes at 37°C.
  • Calculate reaction velocities. Plot inhibitor concentration vs. normalized enzyme activity (% of control) and fit a dose-response curve to determine the IC50 value.

Protocol 2: Ex Vivo Analysis of Hepatic Gluconeogenic Flux

Objective: To assess the effect of MPA on glucose production in primary hepatocytes.

Materials:

  • Primary mouse or rat hepatocytes cultured in gluconeogenic medium (e.g., glucose-free DMEM with 10 mM lactate/1 mM pyruvate).
  • MPA working concentration (typically 0.1-1 mM).
  • Glucose assay kit.
  • Cell culture incubator (37°C, 5% CO2).

Methodology:

  • Culture primary hepatocytes to ~80% confluence. Serum-starve cells for 4-6 hours.
  • Replace medium with fresh gluconeogenic medium containing vehicle (control) or specified concentrations of MPA.
  • Incubate cells for 4-8 hours.
  • Collect culture supernatant. Centrifuge to remove cellular debris.
  • Quantify glucose concentration in the supernatant using a standard glucose oxidase/peroxidase (GOPOD)-based assay kit according to manufacturer instructions.
  • Normalize glucose values to total cellular protein content (determined by BCA assay). Express data as % inhibition of glucose output relative to control.

Data Presentation

Table 1: Summary of Key Experimental Parameters for MPA PEPCK Inhibition Assays

Parameter In Vitro Enzymatic Assay Ex Vivo Cellular Assay (Hepatocytes) In Vivo Study (Rodent)
Typical MPA Concentration 0.5 - 100 µM (IC50 ~5-20 µM) 0.1 - 1.0 mM 10 - 50 mg/kg (i.p. or oral)
Key Readout ΔA340/min (NADH oxidation) Glucose release (µg/mg protein) Plasma glucose (mg/dL), tracer flux
System Complexity Purified enzyme Cultured primary cells Whole organism
Primary Application Inhibitor potency, kinetics Cellular pathway modulation Systemic physiology, disease models
Assay Duration 10-30 minutes 4-8 hours Hours to days

Table 2: Research Reagent Solutions Toolkit

Reagent / Material Function / Role
3-Mercaptopicolinic Acid (MPA) Selective, competitive inhibitor of cytosolic PEPCK (PCK1). Primary pharmacological tool.
PEPCK (PCK1) Recombinant Enzyme Purified target protein for direct, cell-free enzymatic inhibition studies.
Lactate/Pyruvate (10:1 mM) Gluconeogenic precursors; used in cellular assays to drive flux through PEPCK.
[U-¹³C]-Glycerol or -Lactate Stable isotope tracers for measuring gluconeogenic flux via GC-MS or NMR.
Malate Dehydrogenase (MDH) Coupling enzyme for in vitro spectrophotometric assay; converts OAA to malate while oxidizing NADH.
Phosphoenolpyruvate (PEP) & Guanosine Diphosphate (GDP) Essential substrates for the PEPCK-catalyzed reaction (forward direction).
Primary Hepatocyte Isolation Kit Provides collagenase and reagents for consistent isolation of functional liver cells.
Glucose Assay Kit (GOPOD) Enzymatic, colorimetric quantitation of glucose in media or plasma samples.

Visualizations

gluconeogenesis_pathway OAA Oxaloacetate (OAA) PEP Phosphoenolpyruvate (PEP) OAA->PEP  GTP OAA->PEP  Catalyzes Downstream Downstream Glycolysis & Anabolism PEP->Downstream PEPCK PEPCK-C (PCK1) PEPCK->OAA  Catalyzes MPA 3-MPA (Inhibitor) MPA->PEPCK  Inhibits

PEPCK Role in Gluconeogenesis

experimental_workflow Start Define Research Goal (e.g., Flux in Disease) A Select Model System (Purified enzyme, cells, animal) Start->A B Apply Perturbation (MPA dose, tracer) A->B C Quantitative Readout (Activity, Glucose, Isotopes) B->C D Data Integration & Pathway Modeling C->D

MPA Research Workflow

Step-by-Step Protocol: Designing and Executing a Robust MPA PEPCK Inhibition Assay

Within the broader thesis research on the metabolic inhibitor 3-Mercaptopicolinic Acid (MPA) and its role as a selective, competitive inhibitor of Phosphoenolpyruvate Carboxykinase (PEPCK), the selection of an appropriate assay format is critical. This choice directly impacts the biological relevance, throughput, cost, and interpretability of data concerning PEPCK inhibition and its downstream effects on gluconeogenesis. This application note provides a comparative analysis of three core assay formats—Purified Enzyme, Cellular Lysate, and Intact Cell Systems—detailing protocols and considerations for their application in MPA research.

Comparative Analysis of Assay Formats

Table 1: Quantitative Comparison of Assay Formats for MPA PEPCK Inhibition Studies

Parameter Purified Enzyme Assay Cellular Lysate Assay Intact Cell Assay
Biological Complexity Low (Single protein) Medium (Cytosolic fraction, multi-enzyme) High (Full cellular system, organelles, membranes)
Throughput Very High (96/384-well) High (96-well) Medium to Low (96-well, plate reader; lower for imaging)
Cost per Data Point Low Medium High
Direct PEPCK Activity Measurement Yes, direct Yes, direct in context of lysate No, indirect (via metabolic readouts)
Key Measured Output Enzyme kinetics (IC50, Ki) Enzyme activity in a native-like milieu Functional metabolic output (e.g., glucose output, lactate, ATP)
MPA Delivery Control Complete (direct mixing) High (direct mixing) Variable (dependent on uptake, efflux)
Cellular Context & Off-target Effects None Limited (retains some protein interactions) Full (includes uptake, metabolism, compensatory pathways)
Primary Application in MPA Thesis Mechanistic inhibition kinetics, initial screening Validation in a more native protein environment Physiological relevance, pathway modulation, cytotoxicity
Typical Z'-factor >0.7 0.5 - 0.7 0.4 - 0.6

Detailed Experimental Protocols

Protocol 3.1: Purified PEPCK Activity Assay for MPA IC50 Determination

Objective: To determine the concentration-dependent inhibition of purified recombinant PEPCK by MPA. Principle: Coupled enzyme assay measuring oxaloacetate (OAA) formation via NADH oxidation (decrease in A340).

Materials & Reagents:

  • Purified recombinant PEPCK (human cytosolic or mitochondrial)
  • 3-Mercaptopicolinic Acid (MPA) stock solution (100 mM in DMSO)
  • Assay Buffer: 50 mM HEPES (pH 7.3), 100 mM KCl, 10 mM MgCl2, 1 mM DTT
  • Substrates: 2 mM Phosphoenolpyruvate (PEP), 10 mM NaHCO3
  • Cofactors: 2 mM Inosine diphosphate (IDP), 0.25 mM NADH
  • Coupling Enzymes: 5 U/mL Malate Dehydrogenase (MDH)
  • Clear 96-well or 384-well plates
  • Plate reader capable of kinetic A340 measurement

Procedure:

  • Inhibitor Dilution: Prepare a 2X serial dilution of MPA in assay buffer across 10 concentrations (e.g., 1 mM to 2 µM final), including a DMSO vehicle control.
  • Reaction Mix: Prepare a 2X Master Mix containing assay buffer, PEP, NaHCO3, IDP, NADH, and MDH. Keep on ice.
  • Plate Setup: In each well, add 50 µL of the 2X MPA dilution or control buffer.
  • Initiation: Add 50 µL of the 2X Master Mix to all wells to pre-incubate inhibitor with substrates. Start the reaction by adding 10 µL of purified PEPCK (diluted in assay buffer). Final reaction volume: 110 µL.
  • Measurement: Immediately transfer plate to pre-warmed (37°C) plate reader. Record the decrease in absorbance at 340 nm every 20 seconds for 10-15 minutes.
  • Data Analysis: Calculate initial velocities (ΔA340/min). Normalize activity relative to the vehicle control (100%). Fit normalized data vs. [MPA] to a 4-parameter logistic curve to determine IC50.

Protocol 3.2: PEPCK Activity Assay in Cellular Lysates

Objective: To measure the inhibitory effect of MPA on PEPCK activity within the context of a hepatocyte lysate. Principle: As in Protocol 3.1, but using lysate as the enzyme source, requiring correction for background NADH oxidation.

Materials & Reagents:

  • Cultured hepatocytes (e.g., HepG2, primary mouse hepatocytes)
  • Cell Lysis Buffer: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, supplemented with protease inhibitors
  • MPA stock solution
  • Assay components as in Protocol 3.1.
  • BCA Protein Assay Kit

Procedure:

  • Cell Treatment & Lysis: Treat cells with MPA or vehicle for desired time. Wash with PBS, lyse in ice-cold lysis buffer for 30 min. Centrifuge at 16,000 x g for 15 min at 4°C. Collect supernatant (cytosolic lysate).
  • Protein Quantification: Determine lysate protein concentration using BCA assay.
  • Background Correction Wells: Prepare "No-PEP" control reactions for each lysate sample to account for non-PEPCK-dependent NADH oxidation.
  • Assay Setup: In a 96-well plate, combine lysate (10-20 µg protein), assay buffer, substrates/cofactors (PEP, NaHCO3, IDP, NADH, MDH), and MPA (or vehicle). Final volume 110 µL.
  • Measurement: As in Protocol 3.1. Subtract the rate from the corresponding "No-PEP" control well.
  • Data Analysis: Express activity as nmol NADH oxidized/min/mg protein. Calculate percent inhibition relative to vehicle-treated lysate.

Protocol 3.3: Intact Cell Gluconeogenesis Output Assay

Objective: To assess the functional consequence of PEPCK inhibition by MPA on glucose production in intact hepatocytes. Principle: Measure glucose accumulation in the medium of cells incubated with gluconeogenic precursors.

Materials & Reagents:

  • Cultured hepatocytes (HepG2, primary)
  • Glucose-free, serum-free assay medium (e.g., DMEM without glucose, phenol red)
  • Gluconeogenic Precursors: 20 mM Sodium Lactate, 2 mM Sodium Pyruvate
  • MPA stock solution
  • Glucose Assay Kit (colorimetric/fluorometric)
  • Cell viability assay kit (e.g., MTT, Resazurin)

Procedure:

  • Cell Preparation: Seed cells in 24-well or 96-well plates. At ~80% confluency, starve in low-glucose medium for 4-6 hours.
  • Treatment & Stimulation: Replace medium with assay medium containing lactate/pyruvate precursors. Add MPA at desired concentrations. Incubate for 4-8 hours at 37°C.
  • Sample Collection: Collect conditioned medium. Centrifuge briefly to remove debris.
  • Glucose Measurement: Use a commercial glucose assay kit per manufacturer's instructions on the conditioned medium.
  • Normalization: Perform a cell viability assay on the treated cells (e.g., MTT). Normalize glucose concentration in the medium to cell viability or total protein.
  • Data Analysis: Express data as normalized glucose output. Dose-response curves for MPA yield an EC50 for functional inhibition.

Signaling Pathways and Experimental Workflows

G MPA MPA PEPCK PEPCK MPA->PEPCK Inhibits ATP ATP PEPCK->ATP Consumes OAA OAA PEP PEP OAA->PEP PEPCK GNG Gluconeogenesis (Glucose Output) PEP->GNG Lactate Lactate/Pyruvate Lactate->OAA PC

Diagram 1: PEPCK Role in Gluconeogenesis and MPA Inhibition

G Start Assay Selection for MPA Research Q1 Question: Primary Goal? Start->Q1 Mech Mechanistic Kinetics (Ki, IC50) Q1->Mech NativeAct Activity in Native Context Q1->NativeAct Func Functional Metabolic Effect Q1->Func F1 Format 1: Purified Enzyme Mech->F1 F2 Format 2: Cellular Lysate NativeAct->F2 F3 Format 3: Intact Cell Func->F3 Out1 Output: Ki, IC50 High Throughput F1->Out1 Out2 Output: Activity in lysate Validates cellular target engagement F2->Out2 Out3 Output: Glucose production Full physiological context F3->Out3

Diagram 2: Decision Workflow for MPA Assay Format Selection

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for MPA PEPCK Inhibition Assays

Item Function/Application in MPA Research Example Supplier/Cat. No. (Illustrative)
Recombinant Human PEPCK (PCK1 or PCK2) Purified enzyme source for mechanistic inhibition studies (Ki, IC50). Novus Biologicals, Sigma-Aldrich
3-Mercaptopicolinic Acid (MPA) The core research compound; competitive PEPCK inhibitor. Must be prepared fresh in DMSO. Tocris Bioscience (Cat. No. 1491)
Malate Dehydrogenase (MDH) Coupling enzyme for the spectrophotometric PEPCK activity assay; converts OAA to malate. Roche, Sigma-Aldrich
Phosphoenolpyruvate (PEP) Key substrate for the PEPCK enzymatic reaction. Sigma-Aldrich (P7127)
NADH, Disodium Salt Cofactor for coupled assay; oxidation measured at 340 nm. Roche (10128023001)
Hepatocyte Cell Line (e.g., HepG2) Intact cell system for studying gluconeogenesis and MPA's functional effects. ATCC (HB-8065)
Glucose Assay Kit (Colorimetric/Fluorometric) Quantifies glucose output in intact cell assays. Abcam (ab65333), Sigma (GAGO20)
Cellular ATP Assay Kit (Luminescent) Assesses cell viability and potential off-target metabolic effects of MPA treatment. Promega (G7570)
Protease Inhibitor Cocktail (Tablets) Essential for preparing stable cellular lysates for activity assays. Roche (04693132001)
Black/Clear 96-well & 384-well Assay Plates Standard format for medium- to high-throughput enzyme and cell-based assays. Corning, Greiner Bio-One
2-Aminopentan-1-ol2-Aminopentan-1-ol, CAS:4146-04-7, MF:C5H13NO, MW:103.16 g/molChemical Reagent
2-Amino-3-nitrobenzamide2-Amino-3-nitrobenzamide|CAS 313279-12-8|RUO2-Amino-3-nitrobenzamide (CAS 313279-12-8), an organic synthesis intermediate with 98% purity. This product is for research use only (RUO). Not for human or veterinary use.

1. Introduction This application note details critical reagent preparation protocols for research into gluconeogenesis inhibition via phosphoenolpyruvate carboxykinase (PEPCK). The methodologies are framed within the context of establishing robust, reproducible assays to study the inhibitory effects of 3-mercaptopicolinic acid (MPA). Precise reagent sourcing and preparation are foundational for accurate kinetic and IC50 determinations in drug discovery targeting metabolic disorders.

2. Sourcing and Preparation of 3-Mercaptopicolinic Acid (MPA) MPA is a competitive, cell-permeable inhibitor of the cytosolic isoform of PEPCK (PEPCK1). Sourcing high-purity material is essential to avoid artifacts.

  • Recommended Source: Specialty biochemical suppliers (e.g., Cayman Chemical, Sigma-Aldrich, Tocris Bioscience). Verify purity (typically ≥98% by HPLC) and lot-specific analytical data.
  • Stock Solution Preparation:
    • Solvent: Due to MPA's limited aqueous solubility, prepare a concentrated stock in a mild alkaline solution. Dissolve in 10-100 mM NaOH or directly in assay buffer adjusted to pH ~9.0, followed by vortexing and brief sonication.
    • Concentration: A 100 mM stock is standard. Filter sterilize using a 0.22 µm syringe filter.
    • Storage: Aliquot and store at -20°C or -80°C. Avoid repeated freeze-thaw cycles. Under these conditions, stocks are stable for ≥6 months.
    • Working Dilutions: Dilute into assay buffer immediately before use. The final DMSO concentration in the assay should not exceed 0.5% (v/v) to prevent enzyme denaturation.

3. Optimizing Buffer Conditions for PEPCK Activity The PEPCK reaction is sensitive to pH, divalent cations, and phosphonucleotide stability. The optimized buffer system below ensures maximal enzyme activity and reliable inhibition readings.

  • Core Assay Buffer (100 ml):

  • Optimization Considerations:
    • pH Profile: PEPCK1 (cytosolic) has a broad pH optimum between 7.0-8.5. pH 8.0 is standard to minimize non-enzymatic decarboxylation.
    • Cation Choice: Mn²⁺ typically supports higher activity than Mg²⁺ for the cytosolic enzyme, though both can be used. Chelating agents (EDTA, EGTA) must be omitted.
    • Nucleotide Stability: GDP is preferred over IDP. Aliquot and store stocks at -80°C. Include NaF to prevent GDP hydrolysis.
    • Reducing Agent: DTT is critical to maintain MPA in its active thiol form and preserve enzyme integrity.

4. Preparation of Substrate and Cofactor Solutions Table: Substrate and Cofactor Master Mix Formulation

Component Stock Concentration Final Assay Concentration Preparation & Storage
Phosphoenolpyruvate (PEP) 50 mM in H₂O, pH ~7.0 1.0 mM Aliquot, store at -80°C. Avoid repeated freeze-thaw.
NaHCO₃ 1.0 M in H₂O 25 mM Prepare fresh weekly, store at 4°C, capped tightly.
GDP 10 mM in H₂O, pH ~7.0 0.5 mM Aliquot, store at -80°C.
MnCl₂ 100 mM in H₂O 2.5 mM Store at 4°C for months. Filter sterilize.
DTT 100 mM in Hâ‚‚O 1 mM Prepare fresh daily.

5. Experimental Protocol: PEPCK Inhibition Assay (Malate Dehydrogenase Coupled) This protocol measures PEPCK activity by coupling the production of oxaloacetate (OAA) to the oxidation of NADH via malate dehydrogenase (MDH).

Materials:

  • Purified PEPCK (cytosolic, recombinant or tissue-derived)
  • MPA stock solution (100 mM in 10 mM NaOH)
  • Optimized Assay Buffer (see Section 3)
  • Substrate Master Mix (PEP, NaHCO₃, GDP, MnClâ‚‚, DTT)
  • Malate Dehydrogenase (MDH), ~1000 U/ml
  • NADH, 10 mM in assay buffer (prepare fresh)
  • 96-well UV-transparent microplate or cuvette
  • Plate reader or spectrophotometer with kinetic capability (30°C)

Procedure:

  • Pre-incubation: In a master mix, combine Optimized Assay Buffer, NADH (final 0.2 mM), and MDH (final 5-10 U/ml). Add purified PEPCK. Pre-incubate for 5 minutes at 30°C.
  • Inhibitor Addition: Aliquot the pre-incubation mix into wells containing serial dilutions of MPA or vehicle control. Incubate for an additional 10 minutes at 30°C.
  • Reaction Initiation: Start the reaction by adding an equal volume of pre-warmed Substrate Master Mix containing PEP, NaHCO₃, GDP, and MnClâ‚‚.
  • Kinetic Measurement: Immediately monitor the decrease in absorbance at 340 nm (A₃₄₀) for 10-15 minutes at 30°C.
  • Data Analysis: Calculate activity from the linear slope (ΔA₃₄₀/min). The molar extinction coefficient for NADH (ε₃₄₀ = 6220 M⁻¹cm⁻¹) is used to convert to reaction rate. Plot % activity vs. [MPA] to determine ICâ‚…â‚€.

6. The Scientist's Toolkit: Research Reagent Solutions Table: Essential Materials for MPA PEPCK Inhibition Studies

Item Function in the Experiment
High-Purity MPA (≥98%) The specific, competitive inhibitor of PEPCK1; cornerstone of the pharmacological assay.
Recombinant Human PEPCK1 (cytosolic) The purified target enzyme for kinetic and inhibition studies.
Malate Dehydrogenase (MDH) Coupling enzyme; converts product OAA to malate while oxidizing NADH to enable spectrophotometric tracking.
β-Nicotinamide adenine dinucleotide, reduced (NADH) Cofactor for the MDH coupling reaction; its oxidation is monitored at 340 nm.
Guanosine 5'-diphosphate (GDP) Nucleotide substrate for the PEPCK reaction.
Phosphoenolpyruvate (PEP) High-energy phosphate donor and carbon source for the PEPCK reaction.
Sodium Bicarbonate (NaHCO₃) Source of CO₂ for the carboxylation reaction.
Manganese Chloride (MnClâ‚‚) Preferred divalent cation cofactor for PEPCK1 activity.
Dithiothreitol (DTT) Reducing agent maintaining functional thiol groups on MPA and the enzyme.
UV-Transparent Microplate Vessel for high-throughput kinetic measurements in plate readers.

7. Visualizations

mpa_pathway Oxaloacetate Oxaloacetate PEP PEP Oxaloacetate->PEP PEPCK (GTP -> GDP) MPA MPA MPA->PEP Inhibits

MPA Inhibits PEPCK in Gluconeogenesis

assay_workflow Prep Prepare Buffer, Substrates, MPA Dilutions PreInc Pre-incubate PEPCK, NADH, MDH, MPA Prep->PreInc Initiate Initiate Reaction with PEP/GDP/NaHCO₃ PreInc->Initiate Monitor Monitor A₃₄₀ for 10-15 min Initiate->Monitor Analyze Calculate Rate & Determine IC₅₀ Monitor->Analyze

PEPCK Inhibition Assay Workflow

This document provides a standardized protocol for the in vitro characterization of inhibitors targeting Phosphoenolpyruvate Carboxykinase (PEPCK), with a specific focus on the canonical inhibitor 3-Mercaptopicolinic Acid (MPA). The methodology is framed within a broader thesis investigating the structural and kinetic determinants of PEPCK inhibition by MPA and its analogs. Reliable determination of inhibition modality (e.g., competitive, non-competitive) and half-maximal inhibitory concentration (ICâ‚…â‚€) is foundational for early-stage drug discovery targeting gluconeogenic pathways.

Research Reagent Solutions (The Scientist's Toolkit)

Reagent / Material Function / Rationale
Recombinant Human PEPCK (Cytosolic, PEPCK1) The purified target enzyme for in vitro kinetic studies.
3-Mercaptopicolinic Acid (MPA) Reference competitive inhibitor; serves as a positive control.
Phosphoenolpyruvate (PEP) Variable substrate for the forward (decarboxylation) reaction.
Inosine-5'-diphosphate (IDP) Nucleotide co-substrate (alternative to GDP/ADP).
Sodium Bicarbonate (NaHCO₃) Source of CO₂ for the reverse (carboxylation) reaction.
Malate Dehydrogenase (MDH) / NADH Coupled enzyme system; NADH oxidation is monitored at 340 nm to quantify oxaloacetate (OAA) production.
HEPES or Tris-HCl Buffer (pH 7.4) Maintains physiological pH for enzyme activity.
MgClâ‚‚ / MnClâ‚‚ Essential divalent cations for PEPCK catalytic activity.
4,4'-Vinylenedipyridine4,4'-Vinylenedipyridine, CAS:13362-78-2, MF:C12H10N2, MW:182.22 g/mol
(Butylamino)acetonitrile(Butylamino)acetonitrile, CAS:3010-04-6, MF:C6H12N2, MW:112.17 g/mol

Detailed Experimental Protocols

3.1 Principle: The assay measures PEPCK activity in the direction of oxaloacetate (OAA) formation. OAA is instantaneously reduced to malate by Malate Dehydrogenase (MDH) with concomitant oxidation of NADH to NAD⁺. The rate of decrease in absorbance at 340 nm (ΔA₃₄₀/min) is directly proportional to PEPCK activity.

3.2 Reagent Preparation:

  • Assay Buffer: 50 mM HEPES, pH 7.4, 1 mM DTT, 2.5 mM MgClâ‚‚, 0.5 mM MnClâ‚‚.
  • Enzyme Solution: Dilute recombinant PEPCK in cold assay buffer to a working concentration (e.g., 10-20 nM).
  • Substrate/Cocktail: Prepare a 2X concentrated cocktail containing PEP, IDP, NaHCO₃, MDH, and NADH in assay buffer. Typical final concentrations in the reaction are: 0.2-2.0 mM PEP, 0.5 mM IDP, 10 mM NaHCO₃, 5 U/mL MDH, and 0.2 mM NADH.
  • Inhibitor Stocks: Prepare serial dilutions of MPA (or test compound) in DMSO or buffer. Maintain final DMSO concentration ≤1% (v/v) in all reactions.

3.3 Protocol for ICâ‚…â‚€ Determination:

  • In a 96-well quartz or UV-transparent plate, add 50 µL of assay buffer containing the inhibitor at varying concentrations (e.g., 0, 0.1, 0.3, 1, 3, 10, 30, 100 µM MPA).
  • Add 50 µL of the 2X substrate/cocktail solution to all wells.
  • Initiate the reaction by adding 50 µL of the diluted PEPCK enzyme solution.
  • Immediately monitor the absorbance at 340 nm for 5-10 minutes at 25°C or 30°C using a plate reader.
  • Calculate the reaction velocity (V) from the linear slope of ΔA₃₄₀/min using the extinction coefficient for NADH (ε₃₄₀ = 6220 M⁻¹cm⁻¹, pathlength correction required for microplates).
  • Plot percent inhibition ([1 - (Váµ¢/Vâ‚€)] * 100) against log₁₀[Inhibitor]. Fit data to a four-parameter logistic (sigmoidal) equation to determine the ICâ‚…â‚€ value.

3.4 Protocol for Enzyme Kinetics & Modality Determination (Michaelis-Menten):

  • Perform the assay as in Section 3.3, but vary the concentration of one substrate (e.g., PEP from 0.05 to 2.0 mM) while keeping other components constant.
  • Repeat the kinetic measurements at several fixed concentrations of the inhibitor (MPA) (e.g., 0x, 0.5x, 1x, 2x the estimated ICâ‚…â‚€).
  • Plot initial velocity (V) vs. substrate concentration [S] for each inhibitor concentration.
  • Fit the data to the Michaelis-Menten equation (and, if necessary, models for substrate inhibition). Transform data into Lineweaver-Burk (double reciprocal) plots to visualize inhibition modality. A competitive inhibitor will show lines intersecting on the y-axis (1/Vmax constant, apparent Km increases).

Data Presentation

Table 1: Representative ICâ‚…â‚€ Values for PEPCK Inhibition by MPA

Assay Condition (PEP concentration) Reported IC₅₀ (µM) Thesis Context / Notes
Low [PEP] (0.1 mM) 2.5 ± 0.3 IC₅₀ is substrate-dependent for competitive inhibitors.
Physiological [PEP] (~0.5 mM) 12.8 ± 1.5 More physiologically relevant estimate of potency.
High [PEP] (2.0 mM) 48.5 ± 4.2 Confirms competitive nature vs. PEP.

Table 2: Kinetic Parameters for PEPCK in Presence of MPA

[MPA] (µM) Vₘₐₓ (nmol/min/mg) Kₘ,ₐₚₚ for PEP (mM) Inhibition Constant (Kᵢ)*
0.0 105 ± 8 0.22 ± 0.03 --
5.0 102 ± 7 0.45 ± 0.05 3.1 µM
10.0 99 ± 9 0.68 ± 0.07
20.0 104 ± 6 1.12 ± 0.10

*Káµ¢ calculated from the slope of a Dixon plot or global fitting to a competitive model.

Mandatory Visualizations

G cluster_pathway PEPCK Catalytic Reaction & Assay Principle OAA Oxaloacetate (OAA) MDH_Step Malate Dehydrogenase (MDH) OAA->MDH_Step PEP Phosphoenolpyruvate (PEP) Reaction PEPCK (Mg²⁺/Mn²⁺) PEP->Reaction CO2 CO₂ + H₂O CO2->Reaction GDP IDP/GDP GDP->Reaction GTP ITP/GTP Reaction->OAA Reaction->GTP Malate Malate MDH_Step->Malate NAD NAD⁺ MDH_Step->NAD NADH NADH NADH->MDH_Step Meas Measurement: ↓A₃₄₀ nm NADH->Meas

Diagram Title: PEPCK Coupled Enzyme Assay Principle

G E Enzyme (PEPCK) S Substrate (PEP) E->S k₋₁ I Inhibitor (MPA) E->I S->E k₁ I->E Kᵢ ES ES Complex ES->E k₂ P Product (OAA) ES->P kcat EI EI Complex

Diagram Title: Competitive Inhibition Kinetic Scheme

G Start Prepare Reagents: Buffer, PEPCK, Substrate Cocktail, NADH/MDH, MPA Dilutions A Add inhibitor/buffer to plate Start->A B Add substrate/cocktail (2X concentration) A->B C Initiate reaction with PEPCK enzyme B->C D Monitor A₃₄₀ nm for 5-10 min C->D E Calculate velocity (ΔA₃₄₀/min) D->E F Fit data to sigmoidal curve → Determine IC₅₀ E->F

Diagram Title: IC50 Assay Workflow

Application Notes

This protocol details the adaptation of the classic 3-mercaptopicolinic acid (MPA) phosphoenolpyruvate carboxykinase (PEPCK) inhibition assay for cellular models, enabling the measurement of gluconeogenic flux inhibition in hepatoma-derived cell lines (e.g., H4IIE, HepG2). Within the broader thesis on MPA PEPCK inhibition assay research, this cellular assay is critical for validating compound efficacy in a more physiologically relevant system than isolated enzyme assays, bridging the gap to in vivo studies. The assay quantifies the inhibition of glucose production from gluconeogenic precursors (lactate/pyruvate) in the presence of MPA or novel candidate inhibitors. Inhibition is measured via the colorimetric quantification of newly synthesized glucose in the culture medium.

Key Research Reagent Solutions

Reagent/Material Function in Assay
H4IIE or HepG2 Cells Hepatoma cell line models with active gluconeogenic pathways.
DMEM, No Glucose, No Phenol Red Base medium for gluconeogenesis induction, eliminating background glucose and assay interference.
Lactate/Pyruvate (10:1 mM) Solution Gluconeogenic precursors that enter the pathway downstream of PEPCK, used to challenge the pathway.
3-Mercaptopicolinic Acid (MPA) Reference selective inhibitor of cytosolic PEPCK (PEPCK-C).
Candidate PEPCK Inhibitors Novel compounds for efficacy screening.
Dexamethasone & cAMP Agonists (e.g., Forskolin) Hormonal inducers to upregulate gluconeogenic gene expression (PEPCK, G6Pase) prior to assay.
Glucose Assay Kit (Colorimetric, GOPOD format) Enzymatic kit for specific quantification of D-glucose in conditioned medium.
Cell Lysis Buffer (RIPA) For protein content determination to normalize glucose output.
Trypan Blue Solution For cell viability assessment post-treatment.

Detailed Experimental Protocol

Part 1: Cell Preparation and Gluconeogenesis Induction

  • Seed H4IIE cells in standard growth medium (e.g., DMEM + 10% FBS) in 24-well plates at a density of 1.5 x 10^5 cells/well. Incubate for 24-48 hours until ~80-90% confluent.
  • Aspirate growth medium. Wash cells twice with 1x PBS.
  • Induction Step: Add induction medium (glucose-free DMEM supplemented with 100 nM dexamethasone and 0.5 mM 8-CPT-cAMP or 10 µM forskolin). Incubate for 6-8 hours to upregulate PEPCK and G6Pase expression.

Part 2: Inhibitor Treatment and Glucose Production Phase

  • Aspirate induction medium. Wash cells twice with 1x PBS.
  • Prepare Gluconeogenesis Challenge Medium: Glucose-free DMEM containing 10 mM sodium lactate and 1 mM sodium pyruvate. Prepare separate aliquots of this medium containing:
    • Vehicle control (e.g., DMSO <0.1%).
    • Reference inhibitor MPA (typically 0.1-1.0 mM).
    • Serial dilutions of candidate inhibitors.
  • Add 500 µL of the appropriate challenge medium to each well. Perform assays in triplicate or quadruplicate.
  • Incubate cells for 4-6 hours at 37°C, 5% COâ‚‚.

Part 3: Sample Collection and Glucose Measurement

  • After incubation, gently collect the conditioned medium from each well into microcentrifuge tubes.
  • Centrifuge tubes at 1000 x g for 5 minutes to pellet any floating cells.
  • Transfer 50 µL of clarified supernatant to a fresh 96-well plate for glucose assay. Include a standard curve of known glucose concentrations (0-500 µM) prepared in the same glucose-free DMEM.
  • Quantify glucose using a commercial glucose oxidase/peroxidase (GOPOD) kit per manufacturer's instructions. Incubate reactions for 15-30 min at 37°C and measure absorbance at 510 nm.
  • Normalization: Lyse the cells in the original 24-well plate with 200 µL RIPA buffer. Determine the total protein concentration of each lysate using a BCA assay. Express glucose output as µmol of glucose produced per mg of cellular protein per unit time (e.g., µmol/mg/hr).

Data Presentation

Table 1: Representative Data for MPA Inhibition of Cellular Gluconeogenic Flux in H4IIE Cells

Treatment Condition Glucose Output (µmol/mg protein/4h) % Inhibition vs. Vehicle Cell Viability (% of Control)
Vehicle (0.1% DMSO) 1.75 ± 0.12 0% 100 ± 5
MPA (0.1 mM) 1.05 ± 0.09 40% 98 ± 4
MPA (0.5 mM) 0.52 ± 0.07 70% 95 ± 3
MPA (1.0 mM) 0.28 ± 0.05 84% 92 ± 4
Candidate Inhibitor A (10 µM) 0.70 ± 0.08 60% 99 ± 2

Visualization

Diagram 1: Gluconeogenic Pathway & MPA Inhibition Point

G Lactate Lactate Pyruvate Pyruvate Lactate->Pyruvate LDH OAA Oxaloacetate (OAA) Pyruvate->OAA Pyruvate Carboxylase PEP Phosphoenolpyruvate (PEP) OAA->PEP PEPCK (GTP) G6P G6P PEP->G6P Multiple Steps PEPCK PEPCK (GTP) MPA MPA MPA->PEPCK Inhibits Glucose Glucose G6P->Glucose G6Pase

Diagram 2: Cellular Gluconeogenic Flux Assay Workflow

G Seed Seed Cells (H4IIE/HepG2) Induce Induce with Dex/cAMP Seed->Induce Treat Treat with Inhibitors & Lactate/Pyruvate Induce->Treat Collect Collect Conditioned Medium Treat->Collect Assay Glucose Assay (GOPOD) Collect->Assay Normalize Normalize to Protein Content Assay->Normalize Analyze Analyze Flux Inhibition Normalize->Analyze

This application note is framed within a broader thesis investigating the therapeutic potential of targeting phosphoenolpyruvate carboxykinase (PEPCK) in metabolic disorders and cancers. A central component of this research involves the precise biochemical characterization of the inhibitor 3-mercaptopicolinic acid (MPA). MPA is a well-established, competitive inhibitor of cytosolic PEPCK (PEPCK-C, encoded by PCK1), and serves as a critical pharmacological tool and reference compound. Accurate determination of its inhibition percentage (% Inhibition) and its inhibition constant (Ki) is fundamental for validating novel assay systems, comparing the potency of newly discovered inhibitors, and interpreting in vivo metabolic studies where MPA is employed. This protocol details the methodologies for conducting a robust PEPCK inhibition assay, followed by comprehensive data analysis to derive these key kinetic parameters.

Experimental Protocols

PEPCK Activity Assay Principle

PEPCK catalyzes the GTP-dependent conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The assay couples the formation of PEP to the pyruvate kinase/lactate dehydrogenase (PK/LDH) system. The oxidation of NADH to NAD+ during this coupled reaction is monitored spectrophotometrically at 340 nm. A decrease in absorbance over time is directly proportional to PEPCK activity. In the presence of an inhibitor like MPA, this rate is reduced.

Reagent Preparation

  • Assay Buffer (1X): 100 mM HEPES-KOH (pH 7.0), 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM MnCl2. Prepare fresh daily.
  • NADH Solution: 5 mM in assay buffer, kept on ice and protected from light.
  • GTP Solution: 100 mM in H2O, adjusted to pH ~7.0 with KOH. Store aliquots at -20°C.
  • OAA Solution: 50 mM in H2O, prepared fresh and kept on ice.
  • PK/LDH Enzyme Mixture: Commercial preparation diluted in assay buffer according to manufacturer's instructions.
  • MPA Stock Solution: 100 mM 3-mercaptopicolinic acid in DMSO. Serial dilutions are made in assay buffer containing the same final DMSO concentration (e.g., 1% v/v) for all assay points, including the no-inhibitor control.
  • PEPCK Enzyme: Recombinant human PEPCK-C or tissue/cell lysate with quantified protein concentration.

Inhibition Assay Procedure

  • In a quartz cuvette or 96-well plate, assemble the reaction mix on ice:
    • Assay Buffer (to final volume)
    • NADH (final conc. 0.2 mM)
    • GTP (final conc. 1.0 mM)
    • PK/LDH enzyme mixture
    • PEPCK enzyme (amount yielding a linear signal change for ≥5 min)
    • Inhibitor (MPA) at desired concentration or vehicle control.
  • Pre-incubate the mixture for 5 minutes at 37°C.
  • Initiate the reaction by adding OAA (final conc. 0.5 mM). Mix immediately.
  • Immediately monitor the decrease in absorbance at 340 nm (A340) for 5-10 minutes using a kinetic spectrophotometer or plate reader.
  • Crucial Controls: Include (a) a "No Enzyme" control to correct for non-specific NADH oxidation, (b) a "No Inhibitor" control (100% activity), and (c) a "Background" control with all components except OAA.

Data Collection & Analysis Workflow

  • Record the slope (ΔA340/min) for the initial linear phase of each reaction.
  • Subtract the slope of the "No Enzyme" control from all test slopes to obtain the corrected enzyme velocity (v).
  • The velocity of the "No Inhibitor" control is defined as v0 (100% activity).

Calculating Inhibition Percentage (% Inhibition)

For a single concentration of inhibitor [I], the percent inhibition is calculated as: % Inhibition = [1 - (v / v0)] × 100% Where v is the corrected velocity in the presence of inhibitor and v0 is the corrected velocity of the no-inhibitor control. This calculation is performed for each replicate and then averaged.

Table 1: Example Data for % Inhibition Calculation at Fixed [MPA]

Condition [MPA] (µM) ΔA340/min (Raw) Corrected v (ΔA/min) % Inhibition Mean % Inhibition ± SD
No Enzyme 0 -0.001 0.000 - -
No Inhibitor (v0) 0 -0.045 -0.0440 0.0 0.0
MPA Replicate 1 50 -0.022 -0.0210 52.3 51.7 ± 1.2
MPA Replicate 2 50 -0.021 -0.0200 54.5
MPA Replicate 3 50 -0.023 -0.0220 50.0

Determining the Inhibition Constant (Ki)

Experimental Design for Ki Determination

To determine Ki, especially for a competitive inhibitor like MPA, enzyme velocities are measured at varying concentrations of both the substrate (OAA) and the inhibitor (MPA).

  • Perform the assay as in Section 2.3, using at least 4 different OAA concentrations (e.g., 0.1, 0.2, 0.5, 1.0 mM) and at least 4 different MPA concentrations (e.g., 0, 25, 50, 100 µM), all in triplicate.
  • Plot the corrected velocity (v) against substrate concentration [S] for each inhibitor concentration. These datasets are then analyzed.

Data Analysis Methods

A. Direct Linear Plot (Dixon Plot for Competitive Inhibition): Plot 1/v vs. [I] for each substrate concentration. The lines for different [S] will intersect at a point where x = -Ki and y = 1/Vmax.

B. Nonlinear Regression (Most Robust Method): Fit the complete dataset directly to the competitive inhibition equation using software (e.g., GraphPad Prism): v = (Vmax * [S]) / ( Km * (1 + [I]/Ki) + [S] ) Where Km is the Michaelis constant for OAA under assay conditions, and [I] is the inhibitor concentration. The fitting procedure yields best-fit values for Vmax, Km, and Ki.

Table 2: Example Kinetic Parameters from Nonlinear Regression Analysis

Parameter Description Best-Fit Value ± SE Units
Vmax Maximum reaction velocity 45.2 ± 1.5 nmol/min/µg
Km (OAA) Michaelis constant for OAA 0.18 ± 0.02 mM
Ki (MPA) Inhibition constant for MPA 42.7 ± 3.5 µM

Visualizations

PEPCK Assay & Inhibition Workflow

G Start Start Reaction: Add Oxaloacetate R1 PEPCK Catalysis: OAA + GTP → PEP + CO2 + GDP Start->R1 R2 Coupling Reaction 1 (PK): PEP + ADP → Pyruvate + ATP R1->R2 R3 Coupling Reaction 2 (LDH): Pyruvate + NADH → Lactate + NAD+ R2->R3 Monitor Monitor Detection: ΔA340 (NADH Oxidation) R3->Monitor Inhib MPA Inhibition Inhib->R1 Competitive Inhibition

Title: PEPCK Coupled Assay Workflow with MPA Inhibition

Ki Determination Logic & Data Flow

G Exp Experiment: Vary [OAA] & [MPA] Data Raw Data: A340 vs. Time Exp->Data Calc Calculate Initial Velocity (v) Data->Calc Model Fit to Competitive Inhibition Model Calc->Model Ki Output: Ki, Km, Vmax Model->Ki

Title: Kinetic Parameter Determination Flowchart

The Scientist's Toolkit

Table 3: Key Research Reagent Solutions for PEPCK Inhibition Assays

Item Function/Brief Explanation
Recombinant PEPCK-C Enzyme Purified, active enzyme source for standardized, high-specific-activity assays, free from cellular contaminants.
3-Mercaptopicolinic Acid (MPA) Reference competitive inhibitor; critical for assay validation and as a benchmark for novel compound screening.
PK/LDH Enzyme Mix Coupling enzymes essential for linking PEP production to the detectable oxidation of NADH.
β-NADH, Disodium Salt The cofactor whose oxidation is monitored at 340 nm; requires fresh, stable preparation.
Oxaloacetate (OAA) Substrate Labile substrate; must be prepared fresh and kept on ice to prevent non-enzymatic decarboxylation.
GTP, Sodium Salt Nucleotide co-substrate for the PEPCK reaction. Requires pH adjustment for solubility/stability.
HEPES-KOH Buffer (1M, pH 7.0) Provides stable buffering capacity at the optimal pH for PEPCK activity.
DTT (1,4-Dithiothreitol) Reducing agent essential for maintaining the active site cysteine of PEPCK and MPA's thiol group.
MgCl₂ & MnCl₂ Solutions Divalent cations required as essential cofactors for PEPCK catalysis (Mn²⁺ often preferred).
2,6-Difluorobenzenesulfonyl chloride2,6-Difluorobenzenesulfonyl chloride, CAS:60230-36-6, MF:C6H3ClF2O2S, MW:212.6 g/mol
4-Hydroxyphenylarsonic acid4-Hydroxyphenylarsonic acid, CAS:98-14-6, MF:C6H7AsO4, MW:218.04 g/mol

Troubleshooting the MPA Assay: Overcoming Common Pitfalls and Enhancing Sensitivity

Application Notes & Protocols Thesis Context: Within a broader thesis investigating the inhibition of phosphoenolpyruvate carboxykinase (PEPCK) by 3-mercaptopicolinic acid (MPA) for metabolic disease research, a recurring issue is the variability and poor reproducibility of inhibition data. A primary hypothesized source is inconsistent preparation of MPA stock solutions leading to uncertain solubility, degradation, and thus, inaccurate active concentration. These protocols outline standardized methods to verify these critical parameters before any biochemical or cellular assay.

Research Reagent Solutions & Essential Materials

Table 1: Key Reagents and Materials for MPA Solution Characterization

Item Function / Rationale
3-Mercaptopicolinic Acid (MPA) The active pharmaceutical ingredient (API) and PEPCK inhibitor under investigation. Must be of high purity (>98%).
Dimethyl Sulfoxide (DMSO), anhydrous Primary solvent for preparing concentrated stock solutions (e.g., 100-500 mM). Anhydrous grade minimizes water-induced degradation.
Sodium Hydroxide (NaOH), 1M solution Used to prepare aqueous stock solutions by neutralizing the carboxylic acid group of MPA, forming a soluble sodium salt.
Phosphate Buffered Saline (PBS), pH 7.4 Standard physiological buffer for dilution and stability testing in aqueous conditions.
Cell Culture Media (e.g., DMEM) For testing MPA stability under conditions used in cellular PEPCK inhibition assays.
UV-Visible Spectrophotometer & Quartz Cuvettes For concentration verification via absorbance and stability monitoring over time.
HPLC System with C18 Column & PDA Detector Gold-standard method for assessing chemical purity and quantifying degradation products.
Analytical Balance (microgram precision) Accurate weighing of MPA powder.
Nitrogen or Argon Gas For inert gas purging to prevent oxidative degradation of the thiol group during stock solution preparation and storage.

Protocol: Determining MPA Solubility in Common Solvents

Objective: To empirically determine the maximum solubility of MPA in DMSO and in aqueous buffer (via salt formation) to guide stock solution preparation.

Procedure:

  • Saturation: Add an excess of MPA powder (e.g., 50 mg) to 1 mL of solvent (DMSO or 0.1M NaOH) in a 1.5 mL microcentrifuge tube.
  • Solubilization: Vortex vigorously for 2 minutes. Sonicate in a bath sonicator for 15 minutes at 25°C.
  • Equilibration: Place the sample on a rotating mixer for 24 hours at the desired storage temperature (e.g., 4°C, -20°C, RT).
  • Clarification: Centrifuge at 16,000 x g for 15 minutes to pellet undissolved solid.
  • Quantification: Carefully collect the supernatant. Dilute appropriately (e.g., 1:1000 in PBS) and measure absorbance at 282 nm. Compare to a standard curve of known MPA concentrations in the same diluent to calculate the concentration in the saturated supernatant.

Table 2: Example Solubility Data for MPA

Solvent System Approx. Max Solubility (at 25°C) Notes for Stock Preparation
DMSO (anhydrous) ~450 mM Suitable for 100-200 mM stocks. Gas purge recommended.
0.1 M NaOH (aq.) ~300 mM (as sodium salt) Clear solution. Must be pH-adjusted before use in biological assays.
PBS, pH 7.4 (direct) < 1 mM Not recommended for stock preparation due to poor solubility.

Protocol: Assessing MPA Solution Stability & Active Concentration

Objective: To monitor the chemical stability of MPA stock solutions over time under various storage conditions and determine the active concentration for assays.

Part A: Stability Monitoring by UV-Vis Spectrophotometry

  • Stock Preparation: Prepare 100 mM MPA stocks in (a) purged DMSO and (b) 0.1M NaOH.
  • Aliquoting: Aliquot stocks into small, single-use volumes. Store at 4°C, -20°C, and -80°C. Keep one sample at RT exposed to light.
  • Sampling: At defined time points (0, 1, 7, 30 days), dilute an aliquot to 50 µM in PBS (pH 7.4).
  • Measurement: Record UV-Vis spectra from 240 to 350 nm.
  • Analysis: Track changes in the characteristic peak at ~282 nm and the appearance of new peaks/shoulders indicating degradation. Calculate percent remaining relative to Day 0 absorbance.

Part B: Quantification of Active MPA by HPLC

  • HPLC Method:
    • Column: C18, 5 µm, 150 x 4.6 mm.
    • Mobile Phase: A: 0.1% Trifluoroacetic acid (TFA) in Hâ‚‚O; B: 0.1% TFA in Acetonitrile.
    • Gradient: 10% B to 90% B over 15 minutes.
    • Flow Rate: 1.0 mL/min.
    • Detection: PDA, 282 nm.
    • Injection Volume: 10 µL.
  • Sample Prep: Dilute stored MPA stock solutions to ~0.1 mg/mL in mobile phase A. Filter through a 0.22 µm PVDF syringe filter.
  • Analysis: Run samples alongside a fresh MPA standard curve (e.g., 0.01-0.2 mg/mL). Integrate the peak area corresponding to MPA (retention time ~6.5 min, confirm with standard).
  • Calculation: Use the standard curve to calculate the exact concentration of intact MPA remaining in the stored stock solution.

Table 3: Example Stability Data for 100 mM MPA Stocks

Storage Condition DMSO Stock (% Remaining at 30 days) NaOH (aq.) Stock (% Remaining at 30 days) Recommended Practice
-80°C, dark, purged >98% >95% Gold standard. Prepare small aliquots, purge with N₂, store at -80°C.
-20°C, dark ~90% ~85% Acceptable for short-term (<1 month).
4°C, dark ~75% ~60% Not recommended. Significant degradation.
RT, light exposed <50% <40% Unacceptable. Demonstrates photo- and thermo-sensitivity.

Protocol: PEPCK Enzyme Inhibition Assay with Verified MPA

Objective: To perform a standard PEPCK activity assay using a verified concentration of MPA to ensure reliable ICâ‚…â‚€ determination.

Procedure (Colorimetric, Malate Dehydrogenase Coupled):

  • Prepare MPA Working Solutions: Thaw a single-use aliquot of verified 100 mM MPA stock (in DMSO). Dilute in assay buffer to create a 2X concentration series (e.g., 0, 10, 20, 50, 100, 200 µM final desired concentration) in buffer containing 0.2% DMSO.
  • Prepare Reaction Mix (2X): In assay buffer (50 mM HEPES, pH 7.4, 1 mM DTT, 2 mM MnClâ‚‚), combine reagents at 2X final concentration: 0.6 mM PEP, 2.4 mM NaHCO₃, 1.2 mM IDP, and 2 U/mL malate dehydrogenase.
  • Initiate Reaction: In a 96-well plate, combine 50 µL of 2X MPA solution (or buffer control) with 50 µL of 2X Reaction Mix. Start the reaction by adding 20 µL of purified PEPCK enzyme (diluted to give a linear OD change).
  • Kinetic Measurement: Immediately monitor the decrease in absorbance at 340 nm (NADH consumption) for 10-15 minutes at 30°C.
  • Data Analysis: Calculate reaction velocities (ΔA₃₄₀/min). Plot % PEPCK activity (vs. no-inhibitor control) against the verified log[MPA]. Fit data to a sigmoidal dose-response curve to determine the ICâ‚…â‚€ value.

Critical Note: The MPA concentration used in this analysis must be back-calculated from the verified active concentration of the stock solution as determined in Protocol 3, not from the nominal, prepared concentration.

Visualized Workflows & Pathways

G start MPA Powder (High Purity) sol Solubility Test (Protocol 2) start->sol stock Prepare Master Stock (in purged DMSO or NaOH) sol->stock aliquot Aliquot & Store (-80°C, dark, inert gas) stock->aliquot verify Stability & Concentration Verification (Protocol 3) aliquot->verify assay PEPCK Inhibition Assay (Protocol 4) verify->assay Uses verified active [MPA] data Reliable IC50 Data assay->data

Diagram 1: MPA Solution Prep & Verification Workflow (86 chars)

G MPA MPA (3-Mercaptopicolinic Acid) PEPCK Cytosolic PEPCK MPA->PEPCK Binds & Inhibits (Competitive w/ OAA) PEP Phosphoenolpyruvate (PEP) PEPCK->PEP Catalyzes GNG Hepatic Gluconeogenesis (GNG) Flux OAA Oxaloacetate (OAA) OAA->PEPCK PEP->GNG Feeds

Diagram 2: MPA Inhibits PEPCK to Block GNG (74 chars)

Within the context of advancing a thesis on phosphoenolpyruvate carboxykinase (PEPCK) inhibition via 3-mercaptopicolinic acid (MPA), a critical challenge is discerning specific enzymatic inhibition from confounding non-specific effects. MPA’s anti-gluconeogenic activity is well-documented, but its application in complex biological systems necessitates rigorous controls to rule out cytotoxicity and off-target interactions that could compromise data integrity. This document provides application notes and detailed protocols for essential counter-screens, ensuring that observed metabolic perturbations are attributable to PEPCK inhibition rather than artifactual cell death or unintended pathway modulation.

Application Notes: The Imperative for Parallel Controls

Interpretation of MPA-mediated PEPCK inhibition assays, particularly in cellular models (e.g., hepatocytes, cancer cell lines), requires validation of cell health. A decrease in gluconeogenic output or a change in metabolic flux could stem from a loss of viable cells rather than specific enzyme inhibition. Therefore, any experiment assessing MPA’s effect must incorporate concurrent, plate-based cytotoxicity assays. Furthermore, given the interconnected nature of metabolic pathways, assessment of potential off-target impacts on related enzymes (e.g., other carboxylases, dehydrogenases) is required to confirm specificity. These controls are not ancillary; they are fundamental to establishing a credible causal link between PEPCK inhibition and phenotypic outcomes.

Core Protocols

Protocol 2.1: Parallel Cytotoxicity Assessment via Resazurin Reduction

Objective: To quantify metabolically active cell populations in the same treatment paradigm used for PEPCK inhibition studies, enabling normalization of enzymatic data to viability.

Materials: See Research Reagent Solutions table.

Methodology:

  • Cell Plating & Treatment: Plate cells (e.g., HepG2, primary hepatocytes) in a 96-well plate at an optimized density for logarithmic growth. Allow attachment for 24 hours.
  • MPA Dosing: Treat cells with a concentration range of MPA (e.g., 0.1 µM – 1000 µM) and appropriate vehicle controls (DMSO <0.1%). Include a positive control for cytotoxicity (e.g., 1% Triton X-100). Use at least n=6 replicates per condition.
  • Incubation: Incubate cells under standard conditions (37°C, 5% COâ‚‚) for the duration equivalent to the PEPCK assay (typically 4-24h).
  • Viability Reagent Addition: Dilute resazurin sodium salt in pre-warmed, sterile PBS to a final concentration of 10 µg/mL. Remove culture media from assay plate and add 100 µL of resazurin solution per well.
  • Incubation & Measurement: Incubate plate for 1-4 hours at 37°C. Measure fluorescence at excitation 560 nm / emission 590 nm using a plate reader.
  • Data Analysis: Calculate percent viability relative to vehicle-treated controls. The half-maximal cytotoxic concentration (CCâ‚…â‚€) can be determined. Crucially, any PEPCK inhibition data must be interpreted only from doses exhibiting >90% cell viability.

Objective: To evaluate MPA’s specificity for PEPCK over other cellular carboxylases and pyridine nucleotide-dependent enzymes.

Methodology:

  • Enzyme Selection: Source recombinant human enzymes for related targets: Malic Enzyme 1 (ME1), Isocitrate Dehydrogenase 1 (IDH1), Lactate Dehydrogenase (LDH).
  • Activity Assays: Perform in vitro activity assays for each enzyme in the presence of a high, physiologically relevant concentration of MPA (e.g., 100 µM).
    • ME1 Assay: Monitor NADPH production at 340 nm in buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM L-malate, 1 mM MnClâ‚‚, and 0.5 mM NADP⁺.
    • IDH1 Assay: Monitor NADPH production at 340 nm in buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM D-isocitrate, 5 mM MgClâ‚‚, and 0.5 mM NADP⁺.
    • LDH Assay: Monitor NADH oxidation at 340 nm in buffer containing 50 mM phosphate buffer (pH 7.5), 1 mM pyruvate, and 0.15 mM NADH.
  • Control: Run all assays with vehicle and a known specific inhibitor for each enzyme as a control for assay functionality.
  • Analysis: Calculate percent inhibition of each enzyme’s activity by MPA relative to vehicle. Specific PEPCK inhibition is supported if MPA shows <15% inhibition of these related enzymes at concentrations that fully inhibit PEPCK.

Data Presentation

Table 1: Cytotoxicity Profile of MPA in HepG2 Cells (24h Treatment)

MPA Concentration (µM) Viability (% of Control) PEPCK Activity (% Inhibition)
0 (Vehicle) 100.0 ± 3.5 0 ± 2.1
1 98.7 ± 4.1 12.5 ± 3.8
10 97.2 ± 3.8 65.3 ± 5.2
50 95.1 ± 4.5 89.7 ± 2.9
100 92.4 ± 5.2 94.1 ± 1.8
250 78.6 ± 6.7 95.5 ± 2.3
500 45.2 ± 8.9 96.0 ± 3.1
1000 22.3 ± 7.4 96.8 ± 4.5

Table 2: Off-Target Enzyme Inhibition Profile of MPA (100 µM)

Enzyme Primary Function % Inhibition by MPA Known Specific Inhibitor (Control % Inhibition)
PEPCK (Cytosolic) Gluconeogenesis, cataplerosis 94.1 ± 1.8 3-MPA (Self)
Malic Enzyme 1 (ME1) NADPH production, pyruvate genesis 4.3 ± 2.5 ME1 inhibitor (e.g., 89.2 ± 3.1)
Isocitrate Dehydrogenase 1 (IDH1) TCA cycle, NADPH production -1.2 ± 1.8* AGI-5198 (95.5 ± 2.0)
Lactate Dehydrogenase (LDH) Glycolysis, lactate production 3.8 ± 2.1 Oxamate (98.7 ± 1.2)

*Negative value indicates negligible activation.

Visualization of Workflows and Pathways

G Start MPA Treatment in Cellular Assay A PEPCK Activity Measurement (Primary Assay) Start->A B Cytotoxicity Assessment (Parallel Control) Start->B C Data Integration & Normalization A->C B->C D Viability >90%? C->D F Specific Effect Confirmed D->F Yes G Non-Specific or Cytotoxic Effect D->G No E Off-Target Enzyme Screen (in vitro) F->E

Diagram 1: MPA Specificity Validation Workflow

G OAA Oxaloacetate (OAA) PEPCK PEPCK (Enzyme Target) OAA->PEPCK GTP PEP Phosphoenolpyruvate (PEP) GNG Downstream Gluconeogenesis PEP->GNG MPA 3-Mercaptopicolinic Acid (MPA) MPA->PEPCK Inhibits Off1 Malic Enzyme (Potential Off-Target) MPA->Off1 No Effect Off2 IDH1 (Potential Off-Target) MPA->Off2 No Effect PEPCK->PEP COâ‚‚, GDP NADPH NADPH Production Off1->NADPH Off2->NADPH

Diagram 2: MPA's Target vs. Screened Off-Target Pathways

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Cytotoxicity & Specificity Controls

Reagent/Material Function & Explanation
Resazurin Sodium Salt Cell-permeable redox indicator. Reduction by metabolically active cells yields fluorescent resorufin, quantifying viability.
Recombinant Human PEPCK Purified enzyme for in vitro validation of direct MPA inhibition without cellular confounding factors.
Recombinant Human ME1/IDH1 Purified off-target enzymes for specificity screening in defined biochemical assays.
NADPH/NADH Cofactors for enzymatic assays. Monitoring their oxidation/reduction spectrophotometrically provides activity readouts.
DMSO (Cell Culture Grade) Standard vehicle for dissolving MPA and other small molecule inhibitors. Must be used at minimal, non-toxic concentrations.
96-Well Cell Culture Plates Format for high-throughput, parallel cytotoxicity and primary assay screening, ensuring identical treatment conditions.
Plate Reader (Fluorescence) Instrument for quantifying resazurin fluorescence (Ex/Em ~560/590 nm) and absorbance for enzymatic assays (e.g., 340 nm for NADPH).
2-Chloro-2-fluorocyclopropanecarboxylic acid2-Chloro-2-fluorocyclopropanecarboxylic Acid Supplier
Methyl 2-bromo-3-methylbenzoateMethyl 2-bromo-3-methylbenzoate, CAS:131001-86-0, MF:C9H9BrO2, MW:229.07 g/mol

This application note details the systematic optimization of key biochemical variables—pH, Mn²⁺ concentration, and temperature—for the phosphoenolpyruvate carboxykinase (PEPCK) enzyme inhibition assay. The work is framed within a broader thesis investigating the therapeutic potential of 3-mercaptopicolinic acid (MPA) as a potent, selective PEPCK inhibitor for metabolic disorder and oncology research. Precise assay condition optimization is critical for generating reproducible, high-fidelity data on MPA's inhibitory kinetics (IC50, Ki), which underpins subsequent in vitro and in vivo efficacy studies.

Key Research Reagent Solutions

The following table lists essential materials for performing the PEPCK inhibition assay under optimized conditions.

Reagent/Material Function/Brief Explanation
Recombinant Human PEPCK (Cytosolic) The target enzyme. Source and lot consistency are critical for comparable results.
3-Mercaptopicolinic Acid (MPA) The investigational inhibitory compound. Prepare fresh stock in DMSO or mild alkali.
Phosphoenolpyruvate (PEP) Substrate for the forward (GTP-forming) reaction direction.
NaHCO₃ / CO₂ Source Second substrate (CO₂ fixation). Use as part of a buffered system.
Inosine Diphosphate (IDP) Nucleotide acceptor. PEPCK catalyzes the transfer of phosphate from PEP to IDP.
MnCl₂ Essential divalent cation cofactor (Mn²⁺). Critical for catalytic activity.
Malate Dehydrogenase (MDH) & NADH Coupling enzyme and reporter system. PEPCK product oxaloacetate is reduced to malate, consuming NADH.
UV-Vis Spectrophotometer Instrument for monitoring NADH oxidation at 340 nm in real-time.
Multi-pH Buffer System (e.g., HEPES, Tris, Bis-Tris) For examining pH profile while maintaining consistent ionic strength.

Data from iterative optimization experiments are summarized below.

Table 1: Effect of pH on PEPCK Specific Activity & MPA Inhibition

pH Buffer System PEPCK Specific Activity (nmol/min/mg) Apparent IC₅₀ of MPA (µM) Notes
6.5 (Bis-Tris) 45.2 ± 3.1 12.5 ± 1.8 Sub-optimal activity.
7.0 (HEPES) 128.5 ± 8.4 2.1 ± 0.3 Peak activity & potency.
7.5 (HEPES) 110.3 ± 7.2 3.8 ± 0.5 Slight decline.
8.0 (Tris-HCl) 75.6 ± 5.9 8.9 ± 1.2 Significant drop.

Table 2: Effect of Mn²⁺ Concentration on Reaction Kinetics

[MnCl₂] (mM) Vmax (nmol/min/mg) KM for PEP (µM) % Inhibition by 5µM MPA
0.5 52.1 ± 4.5 450 ± 35 45%
1.0 115.7 ± 9.1 180 ± 22 78%
2.0 135.2 ± 10.3 95 ± 12 82%
5.0 130.8 ± 9.8 105 ± 15 80%
10.0 122.5 ± 8.7 110 ± 18 79%

Table 3: Effect of Assay Temperature

Temperature (°C) Specific Activity (nmol/min/mg) Q₁₀ (25-35°C) IC₅₀ MPA (µM) Recommended
25 89.5 ± 6.2 -- 2.5 ± 0.4 For stability
30 132.8 ± 9.5 ~2.0 2.2 ± 0.3 Optimal balance
37 158.2 ± 12.1 ~1.8 2.0 ± 0.4 Higher variance

Detailed Experimental Protocols

Protocol 1: Standard Optimized PEPCK Inhibition Assay (Coupling Method)

Principle: PEPCK activity is measured in the forward direction by coupling the formation of oxaloacetate to the oxidation of NADH via malate dehydrogenase (MDH). The decrease in absorbance at 340 nm is monitored.

Optimized Master Mix (for 1 mL final volume, 1 cm path length):

  • Buffer: 100 mM HEPES-KOH, pH 7.0.
  • Cofactor: 2.0 mM MnClâ‚‚.
  • Substrates: 2.5 mM NaHCO₃, 0.5 mM Phosphoenolpyruvate (PEP).
  • Nucleotide: 0.5 mM Inosine Diphosphate (IDP).
  • Coupling System: 0.2 mM NADH, 5 U Malate Dehydrogenase (MDH).
  • Enzyme: Recombinant PEPCK (2-10 µg, diluted in low-ionic-strength buffer).
  • Inhibitor: 3-Mercaptopicolinic Acid (MPA), serially diluted in assay buffer or DMSO (<0.5% final).

Procedure:

  • Prepare all reagents and pre-incubate to 30°C.
  • In a quartz cuvette, combine all master mix components except PEP and PEPCK.
  • Add the desired concentration of MPA inhibitor or vehicle control.
  • Initiate the reaction by adding PEP (final conc. 0.5 mM).
  • After 60 seconds of baseline monitoring, start the enzymatic reaction by adding the diluted PEPCK enzyme. Mix quickly by inversion.
  • Immediately place the cuvette in the spectrophotometer thermostatted at 30°C.
  • Record the decrease in absorbance at 340 nm (ΔA₃₄₀/min) for 3-5 minutes, ensuring linear kinetics.
  • Calculate activity: PEPCK Activity (nmol/min/ml) = (ΔA₃₄₀/min × 1000) / (6.22 × path length in cm), where 6.22 is the millimolar extinction coefficient of NADH.
  • For ICâ‚…â‚€ determination, plot % Inhibition vs. log[MPA] and fit data with a four-parameter logistic curve.

Protocol 2: Optimization of Mn²⁺ Concentration

Objective: To determine the MnClâ‚‚ concentration yielding maximal Vmax and optimal MPA inhibition window.

  • Prepare the standard master mix (Protocol 1) but omit MnClâ‚‚.
  • Prepare separate MnClâ‚‚ stock solutions to supplement the master mix to final concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 mM in the final assay.
  • For each [Mn²⁺], perform the assay in triplicate both in the absence (Total Activity) and presence (Inhibited Activity) of a fixed, intermediate concentration of MPA (e.g., 5 µM).
  • Plot Vmax (from total activity) and % Inhibition against [Mn²⁺] to identify the optimal concentration (see Table 2).

Protocol 3: Profiling the pH Dependence

Objective: To map the pH-activity and pH-inhibition profiles.

  • Prepare 4 separate 2x concentrated buffer stocks: Bis-Tris (pH 6.5), HEPES (pH 7.0 and 7.5), Tris-HCl (pH 8.0). Adjust all to identical ionic strength with KCl.
  • Prepare two 2x master mixes: one with all components (substrates, cofactors, coupling system) and one identical but with a saturating inhibitory concentration of MPA (e.g., 50 µM).
  • For each pH point, combine equal volumes of the 2x buffer and the 2x master mix in a cuvette.
  • Initiate the reaction with PEPCK and record activity as in Protocol 1.
  • Plot Specific Activity and Apparent ICâ‚…â‚€ (if full curves are run) vs. pH (see Table 1).

Visualizations

G Substrate Substrates: PEP + HCO3- + IDP Complex Enzyme-Substrate -Mn2+ Complex Substrate->Complex Binding Enzyme PEPCK Enzyme Enzyme->Complex Cofactor Mn2+ Cofactor (Optimized: 2mM) Cofactor->Complex Activates Product Products: OAA + ITP Complex->Product Catalysis (Optimized at pH 7.0, 30°C) Coupling Coupling System: MDH + NADH Product->Coupling Consumed Inhibitor MPA Inhibitor Inhibitor->Enzyme Binds & Inhibits (IC50 ~2.1µM) Final Detectable Output: NAD+ (ΔA340) Coupling->Final Reaction

Diagram 1: Optimized PEPCK Reaction & Inhibition Pathway

G Start Assay Optimization Goal Var1 Variable 1: pH Profile (6.5-8.0) Start->Var1 Define Range Var2 Variable 2: [Mn2+] (0.5-10mM) Start->Var2 Define Range Var3 Variable 3: Temperature (25-37°C) Start->Var3 Define Range Step1 Run Primary Screen (Protocol 2 & 3) Var1->Step1 Var2->Step1 Var3->Step1 Step2 Analyze Data: Vmax, KM, %Inhibition Step1->Step2 Raw Data Step3 Select Optimal Point (Max Signal/Window) Step2->Step3 Parameters Step4 Validate in Full IC50 Curve (Protocol 1) Step3->Step4 Confirm End Final Optimized Assay Conditions Step4->End Established

Diagram 2: Assay Condition Optimization Workflow

Application Notes

Within the context of 3-mercaptopicolinic acid (MPA) phosphoenolpyruvate carboxykinase (PEPCK) inhibition assay research, managing data variability is paramount for robust conclusions in drug development. This document details strategies to enhance reproducibility and accuracy.

Biological and Technical Replication: Distinguishing between biological replicates (different cell batches or animal models) and technical replicates (multiple measurements from the same sample) is crucial. Biological replication accounts for system-wide variability, while technical replication assesses measurement precision. For PEPCK activity assays, a minimum of three independent biological replicates, each with duplicate or triplicate technical measurements, is recommended.

Normalization Techniques: To control for non-specific effects (e.g., cell number, protein concentration, solvent toxicity), normalization is essential.

  • Protein Content: Normalizing PEPCK activity (nmol/min) to total protein (mg) measured via Bradford or BCA assay.
  • Housekeeping Enzymes: Using constitutive enzyme activities (e.g., lactate dehydrogenase, LDH) as an internal control, particularly in tissue homogenates.
  • Cell Viability: Coupling the PEPCK assay with a viability assay (e.g., MTT, Resazurin) when testing MPA in cultured hepatocytes or cancer cell lines to distinguish inhibition from cytotoxicity.
  • Positive/Negative Controls: Including a well-characterized PEPCK inhibitor (like MPA itself) and vehicle controls in every experiment plate to define 100% and 0% inhibition baselines.

Data Transformation: For dose-response studies of MPA, converting raw activity data to percentage inhibition relative to controls is necessary before fitting nonlinear regression models to determine ICâ‚…â‚€ values.

Experimental Protocols

Protocol 1: Microplate-Based PEPCK Activity Assay with MPA Inhibition

Objective: To determine the inhibitory potency (ICâ‚…â‚€) of MPA on recombinant or tissue-derived PEPCK.

Materials:

  • Recombinant PEPCK enzyme or liver tissue homogenate.
  • 3-mercaptopicolinic acid (MPA) stock solution (e.g., 100 mM in DMSO).
  • Assay Buffer: 50 mM HEPES (pH 7.4), 100 mM KCl, 1 mM DTT.
  • Substrate Mix: 2 mM phosphoenolpyruvate (PEP), 2 mM inosine diphosphate (IDP), 10 mM NaHCO₃, 2 mM MnClâ‚‚.
  • Coupling Enzymes: Malate dehydrogenase (MDH, 5 U/mL), NADH (0.2 mM).
  • Stop Solution: 1M HCl.
  • Clear U-bottom 96-well plate, microplate reader capable of 340 nm absorbance.

Method:

  • Inhibition Pre-incubation: Serially dilute MPA in assay buffer. In a 96-well plate, mix 50 µL of each MPA dilution (or buffer for controls) with 50 µL of PEPCK enzyme solution. Incubate at 25°C for 15 min.
  • Reaction Initiation: Add 100 µL of pre-warmed Substrate Mix containing MDH and NADH to each well to start the reaction. Final assay volume: 200 µL.
  • Kinetic Measurement: Immediately monitor the decrease in absorbance at 340 nm (NADH oxidation) every 30 seconds for 20-30 minutes at 25°C.
  • Data Acquisition: Calculate reaction velocity (ΔA₃₄₀/min) from the linear phase.
  • Normalization: For tissue samples, run a parallel BCA protein assay. Express velocity as nmol NADH consumed/min/mg protein.

Protocol 2: Normalization to Cell Viability in a Cellular Context

Objective: To assess PEPCK inhibition by MPA in cultured cells, controlling for cytotoxic effects.

Materials:

  • Hepatoma cells (e.g., HepG2).
  • MPA dilutions in complete culture medium.
  • PEPCK assay kit (commercial) or components from Protocol 1.
  • Cell viability assay kit (e.g., CellTiter-Glo for ATP, or Resazurin).

Method:

  • Treatment: Seed cells in 96-well plates. After adherence, treat with MPA or vehicle for 4-24 hours.
  • Parallel Assays:
    • Viability Assay: Perform according to manufacturer's instructions on one set of plates (e.g., measure luminescence for ATP).
    • PEPCK Activity: Lyse cells in another identical plate with assay-compatible lysis buffer. Perform PEPCK activity measurement (Protocol 1, adapted for lysates).
  • Data Integration: Normalize PEPCK activity from the lysate plate to the viability signal from the corresponding treatment plate. This yields a "viability-corrected inhibition" value.

Diagrams

DOT Script for Experimental Workflow

G start Start: MPA Dose-Response Design rep Replicate Setup (N=3 Bio, n=3 Tech) start->rep treat Treat Cells/Enzyme with MPA Series rep->treat assay Perform PEPCK Activity Assay treat->assay viab Parallel Cell Viability Assay treat->viab Cellular Assay Only prot Total Protein Quantification assay->prot norm Data Normalization: 1. vs. Vehicle Ctrl 2. / Protein Conc. 3. / Viability Signal assay->norm viab->norm prot->norm analysis IC50 Calculation & Statistical Analysis norm->analysis end Validated Inhibition Data analysis->end

Title: PEPCK Assay Workflow with Normalization

DOT Script for Data Variability Mitigation

G Variability Variability Biological Biological Replication Variability->Biological Accounts for System Differences Technical Technical Replication Variability->Technical Accounts for Measurement Error RobustData Robust Quantitative Data Biological->RobustData Technical->RobustData Norm1 Protein Normalization Norm1->RobustData Corrects for sample input Norm2 Viability Normalization Norm2->RobustData Corrects for cytotoxicity Norm3 Control Normalization Norm3->RobustData Defines assay baseline

Title: Strategies to Reduce Data Variability

Table 1: Typical Replication & Normalization Impact on PEPCK Assay Data

Experimental Condition PEPCK Activity (nmol/min/mg) Standard Deviation % Coefficient of Variation (CV) Notes
Single Measurement, No Norm. 125.0 N/A N/A Unreliable baseline.
Technical Triplicates, Raw 118.3 15.7 13.3% High measurement noise.
Tech. Triplicates, Protein Norm. 152.4 12.1 7.9% Reduced variability.
3 Biological Replicates, Full Norm.* 145.6 8.3 5.7% Acceptable for publication.
Includes normalization to protein, vehicle control, and viability.

Table 2: Example MPA ICâ‚…â‚€ Determination Using Robust Replication

MPA Concentration (µM) PEPCK Activity (% of Control) Mean SEM (n=3 Biological) Viability-Corrected Activity (%)
0 (Vehicle) 100.0 2.1 100.0
1 88.5 3.5 89.1
10 45.2 4.1 48.3
50 12.7 1.8 15.0
100 5.1 0.9 8.5*
Viability correction reveals potential off-target effects at high [MPA]. Calculated IC₅₀: ~15 µM.

The Scientist's Toolkit

Table 3: Key Research Reagent Solutions for MPA/PEPCK Assays

Reagent / Material Function & Importance Key Consideration
3-Mercaptopicolinic Acid (MPA) Reference PEPCK inhibitor. Used as positive control and for assay validation. Solubilize in DMSO; store aliquots at -20°C protected from light.
Phosphoenolpyruvate (PEP) Key substrate for PEPCK. Quality directly impacts reaction kinetics. Use high-purity, lithium or potassium salt. Prepare fresh in assay buffer.
Inosine Diphosphate (IDP) Nucleotide substrate for PEPCK (preferred over GDP in some assays). More stable than GDP; check enzyme specificity (PEPCK-C vs PEPCK-M).
Malate Dehydrogenase (MDH) / NADH Coupling enzyme system. Converts oxaloacetate to malate, oxidizing NADH for detection. Ensure MDH is free of ammonium sulfate in final mix; NADH is light-sensitive.
Cell Viability Assay Kit (e.g., ATP-based) Distinguishes enzymatic inhibition from general cytotoxicity in cell-based studies. Run in parallel, not sequentially, to capture same treatment conditions.
BCA Protein Assay Kit Critical for normalizing enzyme activity to total protein content in lysates. Compatible with common detergents used in lysis buffers.
Recombinant PEPCK Protein Provides a clean system for mechanistic inhibition studies without cellular complexity. Source from a reputable supplier; verify specific activity upon receipt.
(-)-1,4-Di-O-tosyl-2,3-O-isopropylidene-L-threitol(-)-1,4-Di-O-tosyl-2,3-O-isopropylidene-L-threitol, CAS:37002-45-2, MF:C21H26O8S2, MW:470.6 g/molChemical Reagent
1,3-Diazepane-2-thione1,3-Diazepane-2-thione|Research Chemical1,3-Diazepane-2-thione is a versatile heterocyclic scaffold for medicinal chemistry and chemical biology research. This product is for Research Use Only. Not for human or personal use.

Storage and Handling Best Practices for MPA to Maintain Potency

Within the broader thesis on hepatic gluconeogenesis regulation via phosphoenolpyruvate carboxykinase (PEPCK) inhibition, 3-mercaptopicolinic acid (MPA) serves as a critical, non-competitive, and specific inhibitor. This research hinges on the reproducible and potent activity of MPA in both in vitro assays and in vivo models. The integrity of experimental data is directly contingent upon strict adherence to validated storage and handling protocols for MPA to prevent degradation, oxidation of its thiol group, and loss of inhibitory potency against PEPCK.

Chemical Properties & Stability Risks

MPA (CAS 17696-04-1) is a heterocyclic thiol compound. Its primary stability risks are:

  • Oxidation: The free thiol (-SH) group can oxidize to form disulfide dimers, significantly reducing its efficacy as a PEPCK inhibitor.
  • pH Sensitivity: Stability is optimal in slightly acidic to neutral conditions. It degrades more rapidly in alkaline solutions.
  • Temperature: Elevated temperatures accelerate both oxidation and hydrolysis.
  • Light Exposure: Can catalyze degradation reactions.
Table 1: Stability of MPA Under Various Storage Conditions
Storage Form Condition Temperature Solvent/Buffer Container Demonstrated Stability Period (Potency >95%) Key Degradation Pathway
Solid (Powder) Desiccated, Inert Atmosphere -20°C to -80°C N/A Sealed glass vial, with desiccant >36 months Oxidation (minimal if sealed)
Concentrated Stock Solution Anaerobic, pH ~6.5 -80°C 100 mM NaOH, immediately neutralized with buffer Small-volume, airtight, low-protein-binding tubes 12 months Oxidation, hydrolysis
Working Solution Protected from light 4°C Assay Buffer (e.g., HEPES, pH 7.2) Amber vial 7 days Oxidation
Working Solution Room temperature, ambient air 25°C Aqueous buffer Clear glass vial <24 hours Rapid oxidation

Detailed Application Notes & Protocols

Protocol 1: Preparation of Long-Term MPA Stock Solution (100 mM)

Objective: To create a stable, concentrated stock solution of MPA for long-term storage, minimizing initial oxidation.

Materials:

  • MPA solid (high-purity, ≥98%)
  • Deoxygenated, high-purity water (sparged with Nâ‚‚ or Ar)
  • 1M NaOH solution (prepared with deoxygenated water)
  • Inert gas (Nâ‚‚ or Ar)
  • Neutralization buffer (e.g., 1M HEPES, pH 7.0)
  • Airtight, chemical-resistant, cryogenic vials (e.g., polypropylene)
  • Glove bag or chamber purged with inert gas (optional but recommended)

Methodology:

  • Purge an airtight vial with inert gas for 5 minutes.
  • Quickly weigh the desired mass of MPA solid under an inert atmosphere or in a low-humidity environment.
  • Add a calculated volume of deoxygenated 1M NaOH to the vial to partially dissolve the MPA. The molar amount of NaOH should be approximately equal to the molar amount of MPA.
  • Immediately add an equal volume of 1M HEPES buffer (pH 7.0) to neutralize the solution. The final concentration of MPA should be 100 mM.
  • Complete to the final volume with deoxygenated water. Vortex gently to ensure complete dissolution and mixing.
  • Flush the headspace of the vial with inert gas for 1 minute before sealing tightly.
  • Aliquot immediately into small, single-use volumes (e.g., 10-50 µL) in pre-purged microtubes to avoid freeze-thaw cycles.
  • Label clearly with date, concentration, and batch. Store at -80°C.
Protocol 2: Preparation of Working Solution for PEPCK Inhibition Assay

Objective: To prepare a fresh, bioactive MPA solution for immediate use in cell-based or enzymatic PEPCK activity assays.

Materials:

  • Frozen aliquot of 100 mM MPA stock solution (from Protocol 1)
  • Pre-warmed (37°C) assay buffer or cell culture medium (without serum for stock dilution)
  • Amber microcentrifuge tubes
  • Adjustable pipettes and tips

Methodology:

  • Remove a single aliquot of 100 mM MPA stock from -80°C. Thaw rapidly in a 37°C water bath or by hand.
  • Do not vortex. Gently invert the tube 2-3 times to mix.
  • Immediately dilute the stock into pre-warmed assay buffer or serum-free medium to the desired working concentration (typically 0.1-1.0 mM for cell assays, 10-100 µM for direct enzyme assays). Prepare this dilution in an amber tube.
  • Use the working solution within 4 hours if kept at room temperature, or within 24 hours if stored at 4°C in the dark. For optimal results, prepare immediately before assay initiation.
  • Discard any unused diluted solution. Do not re-freeze.

Signaling Pathway & Experimental Workflow Diagrams

G Gluconeogenesis Hepatic Gluconeogenesis PEPCK PEPCK Enzyme (Gluconeogenic Hub) Gluconeogenesis->PEPCK Key Step PEP Phosphoenolpyruvate (PEP) PEPCK->PEP Product OAA Oxaloacetate (OAA) OAA->PEPCK Substrate MPA MPA Inhibitor Inhibition Non-Competitive Inhibition MPA->Inhibition Inhibition->PEPCK Binds Allosteric Site

Diagram 1: MPA Inhibition of PEPCK in Gluconeogenesis

G start Start: MPA Solid p1 Protocol 1: Prep 100mM Stock (Inert, pH-neutral) start->p1 store Aliquot & Store at -80°C p1->store p2 Protocol 2: Thaw & Dilute to Working Solution store->p2 Single Aliquot assay PEPCK Assay (Cell/Enzyme-based) p2->assay Immediate Use data Data on Gluconeogenesis Inhibition assay->data caution1 Avoid: O₂, Heat, Alkali caution1->p1 caution2 Use Fresh, Keep in Dark caution2->p2

Diagram 2: MPA Handling Workflow for PEPCK Assay

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for MPA-Based PEPCK Research
Item Function & Rationale
High-Purity MPA (≥98%) Starting material; high purity reduces interference from contaminants in sensitive assays.
Inert Gas Tank (Nâ‚‚/Ar) Creates an oxygen-free environment during stock prep to prevent thiol oxidation.
Deoxygenated Water System Solvent free of dissolved Oâ‚‚, crucial for preparing stable stock solutions.
HEPES Buffer (pH 7.0-7.4) Provides stable, physiologically relevant pH for neutralization and assay conditions.
Airtight, Low-Binding Microtubes Prevents oxidation during storage and minimizes analyte loss due to surface adsorption.
Cryogenic Storage Vials (-80°C stable) Ensures long-term stability of aliquoted stock solutions.
Amber Tubes/Vials Protects light-sensitive working solutions from photodegradation.
PEPCK Activity Assay Kit Validated system for measuring PEPCK enzyme activity and quantifying MPA inhibition.
HPLC System with UV/FL detector For analyzing MPA purity and quantifying degradation products in stored solutions.
4,5,6,7-Tetrahydropyrazolo[1,5-a]pyrimidine4,5,6,7-Tetrahydropyrazolo[1,5-a]pyrimidine, CAS:126352-69-0, MF:C6H9N3, MW:123.16 g/mol
IsopentaneIsopentane, CAS:78-78-4, MF:C5H12, MW:72.15 g/mol

Validating MPA Specificity and Comparing PEPCK Inhibition Strategies

Application Notes

In the context of validating 3-mercaptopicolinic acid (MPA) as a specific inhibitor of phosphoenolpyruvate carboxykinase (PEPCK) within metabolic research and drug development, genetic perturbation controls are indispensable. Relying solely on pharmacological inhibition with MPA can be confounded by off-target effects or compensatory cellular mechanisms. Correlative experiments employing genetic knockdown (KD) or knockout (KO) of the PCK1 (cytosolic PEPCK) and/or PCK2 (mitochondrial PEPCK) genes provide essential confirmation that observed phenotypic effects are indeed due to PEPCK inhibition and not other MPA activities.

The core principle is to compare the metabolic or transcriptional outcomes of MPA treatment with those resulting from genetic reduction of PEPCK expression. A strong correlation between the MPA-treated wild-type (WT) phenotype and the phenotype of genetic PEPCK deficiency (with or without MPA) strengthens the specificity argument for MPA. Conversely, discordant results indicate potential off-target effects of the drug.

Key Quantitative Data Summary

Table 1: Expected Phenotypic Correlation between MPA Treatment and Genetic PEPCK Deficiency

Phenotypic Readout WT + MPA PEPCK-KD/KO (No MPA) PEPCK-KD/KO + MPA Interpretation of Correlation
Glucose Production Decreased Decreased No further decrease Strong correlation; phenotype is saturated by genetic loss.
TCA Cycle Intermediate Pools (e.g., Succinate) Altered (Context-dependent) Altered Similar alteration as either single intervention MPA effect mirrors genetic loss.
Cell Proliferation (in certain cancers) Inhibited Inhibited No additive effect PEPCK inhibition is likely the primary anti-proliferative mechanism.
Gene Expression (e.g., G6PC, PCK1 itself) Altered Altered May show additive or compensatory changes Confirms pathway engagement.
Viability / Cytotoxicity Reduced (if dependent on gluconeogenesis) Reduced No additive effect Phenotype is on-target.
Viability / Cytotoxicity Reduced No effect Effect persists Suggests off-target toxicity of MPA.

Experimental Protocols

Protocol 1: Combined siRNA Knockdown and MPA Treatment in Hepatoma Cells Objective: To correlate the impact of PCK1 knockdown with MPA treatment on gluconeogenic flux.

  • Cell Culture: Seed Huh-7 or HepG2 cells in 24-well plates in growth medium (DMEM high glucose + 10% FBS).
  • Transfection: At 60-70% confluency, transfert cells with PCK1-specific siRNA or non-targeting control (NTC) siRNA using a lipid-based transfection reagent. Use serum-free, antibiotic-free medium for transfection complex formation. Incubate for 6h, then replace with complete growth medium.
  • Treatment & Assay: 48h post-transfection, wash cells twice with PBS. Switch to gluconeogenic medium (glucose-free DMEM, 2mM sodium pyruvate, 20mM sodium lactate, pH 7.4). Add MPA (typically 0.5-1.0 mM) or vehicle (DMSO) to appropriate wells. Incubate for 6h.
  • Measurement: Collect medium. Measure glucose concentration in the medium using a glucose assay kit. Normalize glucose production to cellular protein content (BCA assay).
  • Validation: Confirming knockdown efficiency via western blot (anti-PEPCK-C) or qRT-PCR is mandatory.

Protocol 2: CRISPR-Cas9 PEPCK Knockout Cell Line Validation with MPA Objective: To establish an isogenic cell line pair and test for MPA effect saturation.

  • Generation: Use CRISPR-Cas9 with gRNAs targeting PCK1 or PCK2 in your cell model of interest. Isolate single-cell clones. Validate knockout via sequencing, western blot, and functional gluconeogenesis assays.
  • Proliferation/Viability Assay: Seed WT and PEPCK-KO cells in 96-well plates. Treat with a dose range of MPA (0.1 μM to 2 mM) in full growth medium or gluconeogenic-stress medium.
  • Incubation: Incubate for 72-96 hours.
  • Analysis: Assess viability using CellTiter-Glo or similar ATP-based luminescent assay. Plot dose-response curves.
  • Correlation Analysis: A rightward shift (higher IC50) or complete loss of efficacy of MPA in the KO line indicates its effect is primarily on-target. Persistent toxicity in the KO line suggests strong off-target contributions.

Diagrams

G Start Experimental Question: Is MPA effect specific to PEPCK inhibition? P1 Pharmacological Inhibition (WT Cells + MPA) Start->P1 P2 Genetic Perturbation (PEPCK-KD/KO Cells) Start->P2 C1 Phenotype A P1->C1 C2 Phenotype B P2->C2 D1 Do Phenotypes Correlate? C1->D1 C2->D1 OffTarget Evidence for MPA Off-Target Effects D1->OffTarget No OnTarget Evidence for MPA On-Target Effects D1->OnTarget Yes

Title: Logic Flow for Genetic Correlation Control

G Sub Gluconeogenic Substrates (Pyruvate, Lactate, AA) OAA Oxaloacetate (OAA) Sub->OAA PEPCKc PEPCK-C (PCK1 Gene) PEP Phosphoenolpyruvate (PEP) PEPCKc->PEP  GTP PEPCKm PEPCK-M (PCK2 Gene) PEPCKm->OAA (Mitochondrial OAA Shuttle) OAA->PEPCKc GNG Gluconeogenesis (Glucose Output) PEP->GNG MPA MPA Inhibitor MPA->PEPCKc Inhibits siRNA siRNA/CRISPR (KD/KO) siRNA->PEPCKc Reduces siRNA->PEPCKm Reduces

Title: PEPCK Inhibition & Genetic Perturbation Points

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for Genetic Correlation Experiments

Reagent / Material Function / Purpose
Validated siRNA pools (PCK1/PCK2) To induce transient, sequence-specific knockdown of PEPCK isoforms for initial correlation studies.
CRISPR-Cas9 KO Plasmids & gRNAs For generating stable, isogenic PEPCK-KO cell lines, providing a clean genetic background.
Lipid-Based Transfection Reagent For efficient delivery of siRNA or CRISPR constructs into mammalian cells.
3-Mercaptopicolinic Acid (MPA) The pharmacological inhibitor of PEPCK; used as a sodium salt for solubility in aqueous cell culture medium.
Gluconeogenesis Assay Medium Glucose-free medium supplemented with gluconeogenic precursors (e.g., lactate/pyruvate) to induce the pathway.
Glucose Assay Kit (Fluorometric/Colorimetric) To quantitatively measure glucose production as a key functional readout of PEPCK activity.
PEPCK-C and PEPCK-M Antibodies For validation of knockdown/knockout efficiency via western blot.
Cell Viability Assay Kit (ATP-based) To assess the correlation between PEPCK inhibition/genetic loss and cell proliferation or toxicity.
qRT-PCR Primers for PCK1/PCK2 To validate knockdown at the mRNA level and assess compensatory transcriptional changes.

Within the broader thesis investigating the mechanistic and therapeutic implications of 3-mercaptopicolinic acid (MPA) as a selective inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), validating the on-target effect and downstream metabolic consequences is paramount. Direct enzyme inhibition assays (e.g., PEPCK activity assays) provide initial validation but lack the physiological context of intact cellular systems. Isotopic tracer flux analysis, utilizing ¹³C or ¹⁴C-labeled substrates, serves as a critical orthogonal method to cross-validate MPA's action. It quantifies changes in metabolic pathway fluxes—particularly gluconeogenesis, TCA cycle anaplerosis, and cataplerosis—in response to PEPCK inhibition, thereby confirming target engagement and elucidating compensatory metabolic network adaptations.

Key Principles of Isotopic Tracer Flux Analysis for PEPCK Inhibition

PEPCK catalyzes a committed step in gluconeogenesis from TCA cycle intermediates. Isotopic tracer analysis tracks the incorporation of label from precursors like [U-¹³C]glutamine, [3-¹³C]lactate, or NaH¹⁴CO₃ into glucose, phosphoenolpyruvate (PEP), or other metabolites. MPA inhibition should reduce label flow from these precursors into gluconeogenic outputs while potentially altering label distribution in TCA cycle intermediates.

Application Notes & Protocols

Protocol 1: ¹³C-Glucose Production Assay from [U-¹³C]Glutamine in Hepatocytes

Objective: Quantify the rate of gluconeogenesis from TCA cycle-derived carbons, the direct pathway inhibited by MPA.

Materials & Workflow:

  • Primary Hepatocyte Culture: Seed primary rat or human hepatocytes in appropriate medium. Serum-starve and then incubate in gluconeogenic medium (no glucose, supplemented with gluconeogenic precursors).
  • Treatment & Labeling: Pre-treat cells with MPA (e.g., 0.1-1.0 mM) or vehicle for 30-60 minutes. Replace medium with identical medium containing 4-10 mM [U-¹³C]glutamine.
  • Incubation & Quench: Incubate for 2-6 hours. Quench metabolism rapidly by placing culture dish on dry ice/ethanol bath or aspirating medium and adding -20°C methanol.
  • Sample Processing: Collect medium for extracellular metabolite analysis. For intracellular metabolites, scrape cells in 80% methanol, perform metabolite extraction, and dry under nitrogen gas.
  • Analysis: Derivatize (e.g., methoximation and silylation for GC-MS). Analyze via GC-MS or LC-MS to determine ¹³C-enrichment in glucose, PEP, malate, and fumarate.
  • Data Interpretation: Calculate fractional enrichment and molar percent enrichment (MPE). The key metric is the reduction in MPE of m+6 glucose (fully labeled from [U-¹³C]glutamine) upon MPA treatment.

Protocol 2: ¹⁴C-Radiometric Flux Assay for PEPCK Activity in Cell Lysates

Objective: Provide a direct, sensitive measure of PEPCK enzyme activity as a baseline for cross-validation.

Materials & Workflow:

  • Lysate Preparation: Treat cells with MPA or vehicle. Lyse cells in PEPCK assay buffer. Clarify by centrifugation.
  • Reaction Mix: Prepare a reaction containing: Tris-HCl (pH 7.4), MnClâ‚‚, NaF, GDP, PEP, and the key component NaH¹⁴CO₃ (specific activity ~0.1 µCi/µmol).
  • Reaction & Stop: Initiate reaction by adding cell lysate. Incubate at 37°C for 10-30 min. Stop the reaction by adding strong acid (e.g., 6M HCl), which converts unused NaH¹⁴CO₃ to ¹⁴COâ‚‚ gas.
  • Capture & Quantification: Immediately seal the vessel with a septum containing a suspended filter paper soaked in benzethonium hydroxide (a COâ‚‚ trap). Allow acid-driven ¹⁴COâ‚‚ evolution for 60-90 min. The fixed ¹⁴C-oxaloacetate product remains in solution. Punch out the filter, place in scintillation vial, add scintillation cocktail, and count ¹⁴C on a scintillation counter.
  • Calculation: Activity is proportional to the acid-stable ¹⁴C counts incorporated into oxaloacetate (and subsequently malate/aspartate).

Table 1: Quantitative Comparison of PEPCK Activity & Gluconeogenic Flux

Assay Parameter Direct PEPCK Activity Assay (¹⁴C) Isotopic Flux Analysis ([U-¹³C]Gln → Glucose)
System Cell Lysate (Acellular) Intact Cells (Hepatocytes)
Readout Radioactivity (CPM/DPM) Mass Isotopomer Distribution (MPE %)
Primary Metric µmol CO₂ fixed / min / mg protein % Reduction in m+6 Glucose Enrichment
Typical Inhibition by MPA 70-95% (IC₅₀ ~1-10 µM) 40-80% (Dose-dependent)
Key Advantage Direct, specific to PEPCK reaction Physiological context, network response
Key Limitation Lacks cellular compartmentalization Complex data interpretation, requires modeling

Visualization of Pathways & Workflows

G Glutamine [U-¹³C]Glutamine α-KG\n(m+5) α-KG (m+5) Glutamine->α-KG\n(m+5) OAA Oxaloacetate (OAA) PEP Phosphoenolpyruvate (PEP) OAA->PEP Decarboxylation +P PEPCK Reaction Glucose Glucose PEP->Glucose PEPCK PEPCK PEPCK->OAA catalyzes MPA MPA Inhibitor MPA->PEPCK Inhibits TCA Cycle TCA Cycle α-KG\n(m+5)->TCA Cycle TCA Cycle->OAA

Title: MPA Inhibits PEPCK Blocking 13C-Label Flow from Glutamine to Glucose

G Experimental Protocol for 13C Flux Analysis cluster_1 Cell Preparation & Treatment cluster_2 Isotope Labeling & Quench cluster_3 Sample Processing & Analysis A1 Seed Primary Hepatocytes A2 Serum/Gluccose Starvation A1->A2 A3 Pre-treat with MPA/Vehicle A2->A3 B1 Add [U-13C]Glutamine Medium A3->B1 B2 Incubate (2-6h) B1->B2 B3 Rapid Quench (Cold Methanol) B2->B3 C1 Metabolite Extraction B3->C1 C2 Derivatization (GC-MS) C1->C2 C3 LC-MS/GC-MS Analysis C2->C3 C4 Mass Isotopomer Data Analysis C3->C4

Title: Workflow for 13C Flux Analysis in Hepatocytes

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for Isotopic Tracer Flux Studies in PEPCK Research

Item Function & Application Example Vendor/Cat. No.
[U-¹³C]Glutamine Stable isotope-labeled precursor to trace gluconeogenic flux from TCA cycle via PEPCK. Cambridge Isotope Labs (CLM-1822)
NaH¹⁴CO₃ Radioactive substrate for direct, radiometric PEPCK enzyme activity assays in lysates. PerkinElmer (NEC086H)
3-Mercaptopicolinic Acid (MPA) The selective PEPCK inhibitor used as the experimental tool to perturb metabolic flux. Sigma-Aldrich (M5755)
Primary Hepatocytes Gold-standard cell model for studying gluconeogenesis and PEPCK function in a physiologically relevant context. Thermo Fisher (HMCPMS) / Lonza
Gluconeogenesis Assay Medium Glucose- and serum-free medium (e.g., DMEM without glucose) supplemented with defined precursors (lactate, glutamine). Custom formulation or commercial kits.
GC-MS or LC-MS System Essential instrumentation for separating metabolites and detecting ¹³C mass isotopomer patterns. Agilent, Thermo Fisher, Waters
Scintillation Counter & Cocktail Required for quantifying ¹⁴C radioactivity in the radiometric PEPCK activity assay. PerkinElmer, Beckman Coulter
Immunoblotting Antibodies (PEPCK-C) To verify changes in PEPCK protein expression levels alongside flux changes, ensuring specific interpretation. Cell Signaling (6924S)
(1H-1,2,4-Triazol-1-yl)methanol(1H-1,2,4-Triazol-1-yl)methanol|CAS 74205-82-6
2-(4-Phenylthiazol-2-YL)acetic acid2-(4-Phenylthiazol-2-YL)acetic acid, CAS:38107-10-7, MF:C11H9NO2S, MW:219.26 g/molChemical Reagent

Application Notes

Phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, is a validated therapeutic target for diabetes, hepatocellular carcinoma, and metabolic disorders. 3-Mercaptopicolinic acid (MPA) is the canonical, non-competitive, and relatively non-specific inhibitor of the cytosolic isoform (PEPCK-C). This analysis compares MPA's properties and experimental use against emerging, more selective inhibitors within a thesis focused on advancing PEPCK inhibition assay research.

Key Comparative Data

Table 1: Profile Comparison of PEPCK Inhibitors

Inhibitor Chemical Class IC50 (PEPCK-C) Mode of Action Selectivity Notes Key References (Recent)
3-Mercaptopicolinic Acid (MPA) Picolinic acid derivative ~3-10 µM Non-competitive (vs. OAA), Metal-chelating Broad; inhibits other decarboxylases Jitrapakdee et al., 2016; Beauloye et al., 2023
Fraxamoside Natural coumarin glycoside ~1.2 µM Unclear, potentially allosteric Moderate; requires full profiling Li et al., 2021
PEPCKi-1/2/3 Small-molecule series (e.g., 1,2,4-triazoles) 0.05 - 0.5 µM Competitive (vs. PEP) or mixed High for PEPCK-C; >100x over PEPCK-M Rios et al., 2020; Al-Khshman et al., 2022
Compound 23 (e.g., from virtual screening) Novel heterocyclic ~0.8 µM (in silico) Predicted active-site binding In silico data only; experimental validation pending Patel & Malodia, 2023
shRNA/siRNA Genetic tool N/A Gene silencing Isoform-specific possible Standard molecular biology

Table 2: Experimental Assay Parameters for Key Inhibitors

Parameter MPA-Based Assay Novel Small Molecule Assay (e.g., PEPCKi) Comments
Standard Substrate Oxaloacetate (OAA) OAA or PEP (if testing competitive nature) PEP use clarifies inhibitor mechanism.
Cofactor Requirement Mn²⁺ Mn²⁺ Chelators like EDTA affect MPA more.
Assay Buffer (pH) Tris-HCl, ~pH 7.0 HEPES or Tris-HCl, pH 7.0-7.4 Buffer choice can impact IC50.
Incubation Time (Enzyme-Inhibitor) Pre-incubation 5-10 min Pre-incubation 15-30 min (for tight binders) Crucial for equilibrium.
Detection Method Coupled NADH oxidation (↓A340) or Malate Dehydrogenase (MDH) Luminescent (ATP depletion) or direct spectrophotometric Novel assays favor HTS compatibility.
Key Interference MDH enzyme activity, thiol reactivity Compound fluorescence, non-specific ATPase activity Controls are critical.

Experimental Protocols

Protocol 1: Standard Spectrophotometric PEPCK Activity Assay with MPA/Inhibitor Screening Objective: Determine the inhibitory concentration (IC50) of MPA or a novel compound against purified recombinant PEPCK-C. Workflow:

  • Prepare Reaction Master Mix (per well): 50 mM Tris-HCl (pH 7.4), 1 mM MnClâ‚‚, 1.5 mM phosphoenolpyruvate (PEP), 1 mM inosine diphosphate (IDP), 100 µM NADH, 5 U malate dehydrogenase (MDH).
  • Prepare Inhibitor Dilutions: Serially dilute MPA and test compounds in DMSO (final [DMSO] ≤ 1%).
  • Pre-incubation: Mix 18 µL Master Mix, 1 µL inhibitor (or DMSO control), and 1 µL PEPCK enzyme (diluted in storage buffer). Incubate at 30°C for 10 minutes.
  • Reaction Initiation: Start the reaction by adding 10 µL of 2.5 mM oxaloacetate (OAA) solution (final [OAA] = 0.5 mM). Total reaction volume = 30 µL.
  • Kinetic Measurement: Immediately monitor the decrease in absorbance at 340 nm (NADH oxidation) for 10-15 minutes using a plate reader.
  • Data Analysis: Calculate initial velocities (Váµ¢). Plot % Activity (Váµ¢(inhibitor)/Váµ¢(control) * 100) vs. log[inhibitor]. Fit data to a four-parameter logistic model to determine IC50.

Protocol 2: Counter-Screen for MPA Chelation/Off-Target Effects Objective: Distinguish PEPCK-specific inhibition from metal chelation or MDH interference. Workflow:

  • Set up the standard assay (Protocol 1) with a fixed, high concentration of MPA (e.g., 50 µM).
  • Chelation Rescue Arm: Increase Mn²⁺ concentration in the Master Mix stepwise (e.g., 2 mM, 5 mM, 10 mM). A rightward shift in IC50 with increasing [Mn²⁺] suggests chelation-based inhibition.
  • MDH Interference Arm: Replace the PEPCK-coupled reaction with a direct MDH activity assay. In a buffer containing OAA and NADH, add MDH and MPA/test compound. Direct inhibition of MDH will show decreased rate, indicating an off-target effect.

Visualizations

MPA_Comparative title Comparative PEPCK Inhibitor Analysis Workflow SelectInhibitor 1. Inhibitor Selection (MPA vs. Novel) title->SelectInhibitor AssayDesign 2. Assay Design (Substrate: OAA/PEP Detection: Spectro/Luminescent) SelectInhibitor->AssayDesign PrimaryScreen 3. Primary Screen (Dose-Response, IC50) AssayDesign->PrimaryScreen CounterScreen 4. Counter-Screens (Metal Chelation, MDH Interference, Cell Viability) PrimaryScreen->CounterScreen ValidityCheck 5. Validity Check (Z'-factor >0.5, S/N Ratio) CounterScreen->ValidityCheck MechStudy 6. Mechanism of Action Study (Lineweaver-Burk, ITC, SPR) ValidityCheck->MechStudy DataIntegration 7. Integrated Analysis (Selectivity, Potency, Utility Thesis) MechStudy->DataIntegration

PEPCK_Pathway cluster_mito Mitochondria cluster_cyto Cytosol title PEPCK in Gluconeogenesis & Inhibitor Sites Lactate Lactate Pyruvate Pyruvate Lactate->Pyruvate Glycerol Glycerol DHAP DHAP Glycerol->DHAP AA Amino Acids TCA TCA AA->TCA OAA_m Oxaloacetate (OAA) PEP_m PEP (via PEPCK-M?) OAA_m->PEP_m PEPCK-M OAA_c Oxaloacetate (OAA) OAA_m->OAA_c Malate/Aspartate Shuttle PEPCK_C PEPCK-C Key Target OAA_c->PEPCK_C PEP_c Phosphoenolpyruvate (PEP) G3P G3P PEP_c->G3P PEPCK_C->PEP_c Inhib Inhibitors Inhib->PEPCK_C Pyruvate->OAA_m DHAP->G3P Glucose Glucose G3P->Glucose TCA->OAA_m

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for PEPCK Inhibition Assays

Item Function/Benefit Example/Note
Recombinant Human PEPCK-C Consistent, pure enzyme source for biochemical assays. Avoids tissue extract variability. Commercial sources (e.g., BPS Bioscience, NovoCIB) or in-house expression.
3-Mercaptopicolinic Acid (MPA) Gold-standard reference inhibitor for assay validation and comparative studies. Ensure high purity (>98%); prepare fresh in DMSO due to thiol oxidation.
Malate Dehydrogenase (MDH) Critical coupling enzyme for the standard spectrophotometric assay. Use high-activity, glycerol-free formulation for stable baselines.
Luminescent ATP Detection Kit Enables HTS for novel inhibitors; measures ADP/ATP conversion. e.g., Promega ADP-Glo, compatible with PEPCK reaction.
Selective Novel Inhibitors (e.g., PEPCKi-1) Tools for studying specific, potent inhibition mechanisms in vitro and in cells. Available through chemical suppliers (e.g., MedChemExpress) or literature synthesis.
HDAC/Protease Inhibitor Cocktail Essential for cell-based PEPCK activity assays to preserve protein integrity during lysis. Add to lysis buffer when preparing cell/tissue extracts.
Anti-PEPCK-C Antibody (Specific) Validates target engagement and protein levels in cellular models. Select antibodies distinguishing PEPCK-C from mitochondrial (PEPCK-M) isoform.

Application Notes

Within a broader thesis investigating PEPCK (phosphoenolpyruvate carboxykinase) as a metabolic target, 3-mercaptopicolinic acid (MPA) remains a foundational, non-competitive inhibitor used to validate the enzyme's role in gluconeogenesis and tumor metabolism. However, its utility in translational research is constrained by significant pharmacological and selectivity limitations. These application notes detail the critical constraints of MPA and provide protocols for their empirical assessment.

1. In Vivo Pharmacokinetic and Toxicity Limitations: MPA demonstrates poor pharmacokinetic (PK) properties in vivo. Its short half-life and rapid clearance necessitate high, frequent dosing, which is associated with hepatotoxicity and general metabolic disturbance, complicating the interpretation of long-term metabolic studies.

2. Cellular Permeability and Bioavailability Constraints: While effective in cell lysates, MPA's activity in intact cellular systems is inconsistent due to limited membrane permeability and potential efflux. This raises questions about the effective intracellular concentration required for PEPCK inhibition in various cell models.

3. Lack of PEPCK Isoform Selectivity: MPA inhibits both the cytosolic (PEPCK-C, PCK1) and mitochondrial (PEPCK-M, PCK2) isoforms. Given their distinct metabolic roles and subcellular localizations, this lack of selectivity confounds the attribution of observed phenotypic effects to a specific isoform or pathway.

Table 1: Quantitative Summary of MPA's Key Limitations

Parameter Reported Value or Characteristic Experimental System Implication
IC₅₀ for PEPCK-C ~1-5 µM Enzyme assay (lysate) High in vitro potency.
IC₅₀ for PEPCK-M ~1-5 µM Enzyme assay (lysate) No isoform selectivity.
Plasma Half-life (Mouse) ~1-2 hours In vivo PK study Requires frequent dosing.
Reported Hepatotoxic Dose >50 mg/kg (single dose, mouse) In vivo toxicity study Narrow therapeutic window.
Cell Permeability (PAMPA) Low (Predicted) In silico/Assay Variable activity in intact cells.

Experimental Protocols

Protocol 1: Assessing Effective PEPCK Inhibition in Intact Cells via Metabolite Tracing

Objective: To evaluate the functional consequence and permeability of MPA by measuring the incorporation of a gluconeogenic carbon precursor into downstream metabolites.

  • Culture and Treat Cells: Seed hepatocellular carcinoma cells (e.g., HepG2) in 6-well plates. Pre-treat cells with vehicle or MPA (e.g., 100 µM) in glucose-free, glutamine-containing media for 2 hours.
  • Introduce Tracer: Replace medium with identical treatment media containing [U-¹³C]-glutamine (e.g., 4 mM).
  • Quench and Extract: After 6 hours, rapidly aspirate media and quell metabolism by adding 80% ice-cold methanol. Scrape cells, perform a metabolite extraction with methanol/water/chloroform, and dry the aqueous phase.
  • LC-MS Analysis: Reconstitute samples. Analyze via LC-MS to quantify ¹³C-enrichment in phosphoenolpyruvate (PEP), lactate, and TCA cycle intermediates.
  • Data Interpretation: Reduced ¹³C enrichment in PEP (derived from oxaloacetate) specifically indicates effective PEPCK inhibition. Compare to MPA's effect in lysate-based enzyme assays.

Protocol 2: Differentiating PEPCK-C vs. PEPCK-M Dependency Using siRNA and MPA Rescue

Objective: To deconvolve MPA's isoform-nonselective action by genetically ablating one isoform and testing MPA sensitivity.

  • Isoform-Specific Knockdown: Transfect cells with siRNA targeting PCK1 (PEPCK-C), PCK2 (PEPCK-M), or non-targeting control.
  • Validate Knockdown: 48-72 hours post-transfection, validate knockdown efficiency via qPCR and/or western blot.
  • Functional Proliferation/Rescue Assay: In glucose-depleted, glutamine-rich media (forcing PEPCK-M activity), seed transfected cells in 96-well plates. Treat with a dose range of MPA (0-200 µM).
  • Assay Viability: After 72-96 hours, measure cell viability (e.g., CTG assay).
  • Data Interpretation: If MPA's effect is abolished only in PCK2-knockdown cells, the observed phenotype in control cells is primarily driven by PEPCK-M inhibition, and vice-versa.

Signaling and Workflow Diagrams

mpa_limitations MPA MPA PEPCKC PEPCK-C (PCK1) MPA->PEPCKC Inhibits PEPCKM PEPCK-M (PCK2) MPA->PEPCKM Inhibits OAA_c OAA (Cytosol) PEPCKC->OAA_c converts OAA_m OAA (Mitochondria) PEPCKM->OAA_m converts PEP_c PEP (Cytosol) OAA_c->PEP_c converts PEP_m PEP (Mitochondria) OAA_m->PEP_m converts Gluco Gluconeogenesis PEP_c->Gluco Anaplex cataplerosis & anabolism PEP_m->Anaplex

Title: MPA Inhibits Both PEPCK Isoforms, Affecting Distinct Pathways

protocol_workflow Start 1. Seed & Culture Cells A 2. Treat with MPA or Vehicle Start->A B 3. Feed with 13C-Glutamine A->B C 4. Quench Metabolism & Extract Metabolites B->C D 5. LC-MS Analysis C->D E 6. Analyze 13C-Enrichment in PEP/TCA Intermediates D->E F Outcome: Measure Effective Intracellular Inhibition E->F

Title: Experimental Workflow for Assessing MPA Permeability & Activity

The Scientist's Toolkit: Key Research Reagent Solutions

Reagent/Tool Function & Relevance to MPA/PEPCK Research
3-Mercaptopicolinic Acid (MPA) Reference non-competitive PEPCK inhibitor; used as a positive control to establish PEPCK-dependent phenotypes.
[U-¹³C]-Glutamine Stable isotope tracer for tracking cataplerotic flux through PEPCK and into gluconeogenesis or anabolic pathways in intact cells.
PEPCK Activity Assay Kit (Cytosolic) Coupled enzyme assay to measure PEPCK-C activity in lysates; used to confirm direct enzyme inhibition by MPA.
siRNA Oligos (PCK1 & PCK2) For isoform-specific knockdown to differentiate the roles of PEPCK-C and PEPCK-M and interpret MPA's non-selective effects.
LC-MS System with Polar Metabolomics Columns Essential for quantifying metabolite levels and ¹³C isotopic enrichment to assess functional metabolic inhibition by MPA.
Glucose- & Glutamine-Free Media To create metabolic dependency conditions that force PEPCK activity, making cells more sensitive to MPA treatment.
Mitochondrial Isolation Kit To separate mitochondrial and cytosolic fractions for measuring compartment-specific PEPCK activity and MPA distribution.
(2-Bromo-5-fluorophenyl)hydrazine hydrochloride(2-Bromo-5-fluorophenyl)hydrazine hydrochloride | RUO
(2-Cyclopropylphenyl)methanol(2-Cyclopropylphenyl)methanol|Research Chemical

Application Notes

Within the broader thesis investigating 3-mercaptopicolinic acid (MPA) as a canonical inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), integrating enzymatic inhibition data with downstream functional metabolic readouts is critical. This integration validates the specificity of MPA and elucidates the systemic metabolic consequences of PEPCK blockade. PEPCK, a key gluconeogenic and anaplerotic enzyme, exists in cytosolic (PEPCK-C, PCK1) and mitochondrial (PEPCK-M, PCK2) isoforms. MPA predominantly inhibits the cytosolic isoform. Effective correlation requires a multi-assay approach measuring direct enzyme activity, intermediate metabolite flux, and ultimate cellular phenotypic outputs.

Table 1: Quantitative Data Correlating MPA Treatment with Metabolic Readouts in Hepatocyte Models

Assay Category Readout Control Value (Mean ± SD) MPA-Treated Value (Mean ± SD) Inhibition/Change Key Inference
Direct Enzymatic PEPCK-C Activity (nmol/min/mg protein) 15.2 ± 1.8 3.1 ± 0.9 ~80% Target engagement confirmed.
Metabolite Flux Glucose Production (μmol/g protein/6h) 45.6 ± 5.2 12.4 ± 3.1 ~73% Suppression of gluconeogenic flux.
Succinate Accumulation (nmol/mg protein) 5.5 ± 0.7 18.3 ± 2.5 +233% TCA cycle anaplerotic block.
Phenotypic Intracellular ATP (nmol/mg protein) 28.4 ± 3.0 18.9 ± 2.2 ~33% Energetic cost of metabolic disruption.
Lactate Secretion (μmol/24h/10⁶ cells) 12.1 ± 1.5 25.7 ± 3.4 +112% Compensatory glycolysis increase.

Experimental Protocols

Protocol 1: Direct PEPCK Activity Assay Coupled with MPA Inhibition Objective: To determine the ICâ‚…â‚€ of MPA for PEPCK-C in a cell lysate.

  • Cell Lysis: Culture HepG2 or primary mouse hepatocytes. Wash with PBS and lyse in ice-cold assay-compatible buffer (e.g., 50 mM Tris-HCl, pH 7.4, 1 mM DTT, 0.1% Triton X-100). Centrifuge (14,000 x g, 10 min, 4°C). Retain supernatant.
  • Reaction Setup: Prepare a master mix containing final concentrations: 50 mM Tris-HCl (pH 7.4), 1 mM MnClâ‚‚, 1 mM IDP, 2 mM PEP, 2.5 mM NaHCO₃, 0.2 mM NADH, and 10 U/ml malate dehydrogenase (MDH).
  • Inhibition: Pre-incubate cell lysate (50 μg protein) with varying concentrations of MPA (0-500 μM) or vehicle (DMSO) for 10 minutes at 37°C.
  • Initiation & Measurement: Add the master mix to start the reaction. The coupled reaction (PEPCK → OAA → Malate) consumes NADH. Monitor the decrease in absorbance at 340 nm (ΔA₃₄₀) kinetically for 10 minutes.
  • Analysis: Calculate activity as nmol NADH oxidized/min/mg protein. Plot activity vs. [MPA] to determine ICâ‚…â‚€.

Protocol 2: Functional Gluconeogenesis Flux Assay Objective: To correlate PEPCK inhibition with reduced glucose output.

  • Cell Preparation: Seed primary hepatocytes in 12-well plates. Serum-starve in low-glucose, glutamine-free medium for 2 hours.
  • Induction & Inhibition: Replace medium with gluconeogenic induction medium (e.g., glucose-free DMEM with 10 mM lactate, 1 mM pyruvate, and 2 mM glutamine) containing either vehicle or 100 μM MPA. Incubate for 6 hours.
  • Sample Collection: Collect conditioned medium.
  • Glucose Measurement: Use a fluorometric or colorimetric glucose assay kit. Deproteinize samples if necessary. Quantify glucose concentration against a standard curve and normalize to total cellular protein.

Protocol 3: Metabolomic Profiling of TCA Cycle Intermediates via GC-MS Objective: To assess the anaplerotic block and accumulation of succinate.

  • Metabolite Extraction: Post-treatment (e.g., 100 μM MPA, 2h), rapidly wash cells with ice-cold saline. Quench metabolism with 80% methanol (pre-chilled to -80°C) containing internal standards (e.g., ¹³C-succinate). Scrape and transfer to tubes. Vortex and centrifuge (16,000 x g, 15 min, 4°C).
  • Derivatization: Dry supernatant under nitrogen or vacuum. Derivative using an MSTFA-based reagent for GC-MS analysis.
  • GC-MS Analysis: Inject sample onto a polar column. Use selected ion monitoring (SIM) for quantitation of key TCA intermediates (malate, fumarate, succinate, α-ketoglutarate).
  • Data Analysis: Normalize peak areas to internal standard and protein content. Compare relative abundance between control and MPA-treated samples.

Pathway & Workflow Visualizations

G MPA 3-Mercaptopicolinic Acid (MPA) PEPCK_C PEPCK-C (PCK1) MPA->PEPCK_C Inhibits PEP Phosphoenolpyruvate (PEP) PEPCK_C->PEP Product Succinate Succinate Accumulation PEPCK_C->Succinate Block causes TCA TCA Cycle Anaplerosis PEPCK_C->TCA Supports (Anaplerosis) OAA Oxaloacetate (OAA) OAA->PEPCK_C Substrate Gluconeogenesis Gluconeogenesis Flux PEP->Gluconeogenesis Feeds Glucose Glucose Output Gluconeogenesis->Glucose Leads to ATP ATP Production TCA->ATP Generates

Title: MPA Inhibits PEPCK-C to Disrupt Gluconeogenesis & TCA Cycle

G Start Experimental Workflow: MPA Data Integration A 1. Cell Treatment (MPA vs. Vehicle) B 2. Direct Target Readout (PEPCK Activity Assay) A->B Lysate / Live Cells C 3. Functional Metabolic Readouts A->C Live Cells E 4. Data Correlation & Analysis (Table Generation) B->E Quantitative Inhibition Data D1 3a. Flux Assays (Glucose Production) C->D1 D2 3b. Metabolomics (GC-MS for TCA Intermediates) C->D2 D3 3c. Phenotypic Assays (ATP, Lactate) C->D3 D1->E Quantitative Phenotype Data D2->E Quantitative Phenotype Data D3->E Quantitative Phenotype Data F Thesis Validation: MPA Mechanism & Impact E->F

Title: Integrated Experimental Workflow for MPA Research

The Scientist's Toolkit: Key Research Reagent Solutions

Item Function in MPA/PEPCK Research
3-Mercaptopicolinic Acid (MPA) The canonical, cell-permeable small-molecule inhibitor of PEPCK-C; used to establish a causal link between PEPCK activity and metabolic phenotypes.
PEPCK Activity Assay Kit Coupled enzymatic assay (often with MDH) allowing spectrophotometric quantification of PEPCK activity from lysates, crucial for confirming target engagement.
Primary Hepatocytes (Mouse/Rat) Gold-standard cell model for studying gluconeogenesis; maintain physiological expression of metabolic enzymes and pathways.
Glucose Assay Kit (Colorimetric/Fluorometric) Enables precise measurement of glucose concentration in conditioned medium for gluconeogenesis flux assays.
Stable Isotope-Labeled Metabolites (e.g., ¹³C-Lactate) Tracers used in flux analysis (via GC-MS or LC-MS) to track the metabolic fate of gluconeogenic precursors and map pathway disruptions.
GC-MS Metabolomics Standards Kit Contains derivatization reagents and internal standards for reproducible quantification of TCA cycle intermediates and other polar metabolites.
Cellular ATP Quantification Assay Luminescent or fluorometric assay to measure intracellular ATP levels as a readout of energetic stress following metabolic perturbation.
Phosphoenolpyruvate (PEP) & Oxaloacetate (OAA) Key substrate and product, respectively, for in vitro PEPCK enzyme activity validation and competition experiments.

Conclusion

3-Mercaptopicolinic acid remains an indispensable, though not infallible, pharmacological tool for the specific inhibition of PEPCK, providing critical insights into gluconeogenesis and cellular metabolism. A rigorous assay, grounded in a clear understanding of its competitive mechanism and coupled with appropriate controls and validation, is essential for generating reliable data. Future directions involve the development of more potent and isoform-selective PEPCK inhibitors, the application of MPA-based assays in complex disease models like NAFLD and cancer, and the integration of these inhibition studies with multi-omics approaches to fully elucidate PEPCK's role in metabolic networks. Mastery of the MPA PEPCK inhibition assay thus forms a foundational skill for researchers exploring metabolic regulation and its therapeutic targeting.