Measuring Phosphoenolpyruvate Carboxykinase (PEPCK) Activity

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) is a part of the carbon-carbon lyase enzyme superfamily that catalyzes the reversible conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP), with the help of co-substrate guanosine triphosphate (GTP) as a phosphate donor.

Two isoforms of PEPCK are found in humans — a mitochondrial form (PEPCK-M, also known as PCK2), and a cytosolic form (PEPCK-C, also known as PCK1). PEPCK-C (PCK1) is a rate-limiting enzyme in gluconeogenesis.

Current studies have revealed a negative correlation between glycemic control and PEPCK-C activity in db/db diabetic mice. Transgenic mice with overexpression of PEPCK show attenuated insulin signaling, decreased hepatic insulin sensitivity, and increased basal hepatic glucose production.

This article reports a selective enzymatic assay for the analysis of PEPCK activity in different biological samples. The PEPCK activity assay, a high-throughput microplate-based enzymatic assay, utilizes a set of exclusively engineered enzymes that convert carbonate and phosphoenolpyruvate into a series of intermediates, eventually yielding pyruvate, which then reacts with a colorless probe to form a stable chromophore detected by spectrophotometry (λabs = 570 nm). Moreover, the color intensity is directly proportional to the amount of PEPCK activity. The assay is selective, highly sensitive, simple to execute, and has a detection limit of below 10 µU/well of PEPCK activity in different biological samples.

In brief, the Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay Kit serves as a user-friendly and powerful tool for measuring PEPCK activity in biological matrices and purified samples (BioVision, Inc. Cat. # K359-100).

Background

  • PEPCK (EC 4.1.1.32) is a member of the carbon-carbon lyase superfamily and is a rate-controlling enzyme for the gluconeogenesis pathway.
  • PEPCK catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP), utilizing GTP as a phosphate donor.
  • PEPCK has two isoforms: Mitochondrial (PEPCK-M, also known as PCK2) and Cytosolic (PEPCK-C, also known as PCK1).
  • Overexpression of PEPCK-C in mice results in a Type II Diabetes-like phenotype, while adenovirus-directed shRNA knockdown of hepatic PEPCK-C enhanced glucose tolerance and fed glycaemia in the db/db diabetic mouse model (similar to the results of oral metformin treatment)
  • Current studies demonstrate that PEPCK-M is a pro-survival factor that is upregulated in several cancer cells

PEPCK

Principle of PEPCK Enzymatic Assay

Principle of PEPCK Enzymatic Assay

Recombinant Human PEPCK-C Purification and Activity

SDS-PAGE (4-20%) of human recombinant PEPCK-C (A) and reaction kinetics of recombinant PEPCK-C (B).

Figure 1. SDS-PAGE (4-20%) of human recombinant PEPCK-C (A) and reaction kinetics of recombinant PEPCK-C (B).

Recombinant PEPCK-C Enzyme Kinetic Constants

Determination of kinetic constants for metabolism of the substrates phosphoenolpyruvate (A) and guanosine diphosphate (GDP) (B) by PEPCK-C.

Figure 2. Determination of kinetic constants for metabolism of the substrates phosphoenolpyruvate (A) and guanosine diphosphate (GDP) (B) by PEPCK-C.

PEPCK Activity Assay Pyruvate Standard Curve

Pyruvate standard (0 – 10 nmole/well) reaction kinetics (A) and background-subtracted pyruvate standard curve (B). One mole of pyruvate is equivalent to the metabolism of one mole of phosphoenolpyruvate.

Figure 3. Pyruvate standard (0 – 10 nmole/well) reaction kinetics (A) and background-subtracted pyruvate standard curve (B). One mole of pyruvate is equivalent to the metabolism of one mole of phosphoenolpyruvate.

Detection of PEPCK Activity in HEK1 Cell Lysates

Reaction progress curves of PEPCK activity detected in HEK1 cell lysates with (A) or without (B) phosphoenolpyruvate.

Figure 4. Reaction progress curves of PEPCK activity detected in HEK1 cell lysates with (A) or without (B) phosphoenolpyruvate.

Detection of PEPCK Activity in Biological Samples

PEPCK specific activity in biological samples. PEPCK activity was measured in lysates prepared from HEK 293 cells (66 µg protein), rat liver (223 µg protein) and rat kidney (145 µg protein).

Figure 5. PEPCK specific activity in biological samples. PEPCK activity was measured in lysates prepared from HEK 293 cells (66 µg protein), rat liver (223 µg protein) and rat kidney (145 µg protein).

PEPCK Unit Definition: One unit of phosphoenolpyruvate carboxykinase is the amount of enzyme that will produce 1.0 µmol of pyruvate per minute at pH 7.5 at 37 °C.

Applications

  • Analysis of potential PEPCK modulating anti-diabetic compounds
  • Analysis of PEPCK-mediated metabolic pathways such as: glyceroneogenesis, insulin signaling, and gluconeogenesis
  • Measurement of the activity of phosphoenolpyruvate carboxykinase (PEPCK) in biological samples

Conclusion

BioVision’s PEPCK Activity Assay Kit is a useful tool for detecting the activity of PEPCK in biological samples as well as detecting potential PEPCK activity modulators for drug discovery. The assay is sensitive (≤10 µU/sample), easy to perform, and high-throughput adaptable.

References

  • Disregulated Glyceroneogenesis: PCK1 as a candidate diabetes and obesity gene. E.G. Eeale, R.E. Hammer, B. Antoine and C. Forest. Trends in Endocrinology and metabolism. 13:129-135, 2004.
  • Phosphoenolpyruvate Carboxykinase and the critical role of cataplerosis in the control of hepatic metabolism. P. Hakimi, M.T. Johnson, T. Yang, D.F. Lepage, R.A. Conlon, S.C. Kachan, L. Reshef, S.M. Tilghman and R.W.Hanson. Nutrition & Metabolism, 2: 33, 2005.
  • What is the metabolic role of Phosphoenolpyruvate Carboxylase? J. Yang, S.C. Kalhan, and R.W. Hanson. The Journal of Biological Chemistry. 284: 27025-27029, 2009.
  • Mitochondrial Phosphoenolpyruvate Carboxylase (PEPCK-M) is a Pro-survival, Endoplasmic Reticulum (ER) Stress Response Gene involved in Tumor Cell Adaptation to Nutrient Availability. A. mendez-Lucas, P. Hyossova, L. Novellasdemunt, F. Vinals, and J.C. Perales. The Journal of Biological Chemistry. 289: 22090-22102, 2014.

BioVision Incorporated

BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.

BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.


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Last updated: Feb 21, 2020 at 9:30 AM

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