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A High-Throughput Assay to Measure Phosphoenolpyruvate Carboxykinase (PEPCK) Activity in Biological Samples
EP27512
Poster Title: A High-Throughput Assay to Measure Phosphoenolpyruvate Carboxykinase (PEPCK) Activity in Biological Samples
Submitted on 25 Apr 2018
Author(s): Shunan Li, Ph.D., Keith Cheung, Ph.D. Grigoriy Tchaga, Ph.D. Jianping Xu, Ph.D. and Gordon Yan, Ph.D. Acknowledments: Kyle Schmitt, Ph.D • Tanvi Gangakhedkar • Gustavo Chavarria, Ph.D. • Kwang Kim, Ph.D. • Robert Stephenson, Ph.D. • Hong Qu, Ph.D.
Affiliations: BioVision, Inc.
This poster was presented at Experimental Biology 2018
Poster Views: 1,071
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Poster Information
Abstract: Phosphoenolpyruvate Carboxykinase (PEPCK, EC 4.1.1.32) is a member of the carbon-carbon lyase
enzyme superfamily that catalyzes the reversible conversion of oxaloacetate (OAA) into
phosphoenolpyruvate (PEP), using the co-substrate guanosine triphosphate (GTP) as a phosphate
donor. In humans, two isoforms of PEPCK are found: a cytosolic form (PEPCK-C, also called PCK1)
and a mitochondrial form (PEPCK-M, also called PCK2). PEPCK-C (PCK1) is a rate-limiting enzyme
in gluconeogenesis. Recent studies have shown a negative correlation between PEPCK-C activity
and glycemic control in db/db diabetic mice. Transgenic mice with overexpression of PEPCK exhibit
attenuated insulin signaling, increased basal hepatic glucose production and decreased hepatic
insulin sensitivity.
Here, we report a selective enzymatic assay for analysis of PEPCK activity in a variety of biological
samples. The PEPCK activity assay is a high-throughput microplate-based enzymatic assay that
utilizes a set of specifically engineered enzymes that convert phosphoenolpyruvate and carbonate
into a series of intermediates, ultimately yielding pyruvate, which subsequently reacts with a colorless
probe to form a stable chromophore detected by spectrophotometry (λabs = 570 nm). The color
intensity is directly proportional to the amount of PEPCK activity. The assay is simple to perform,
selective and highly sensitive, with a detection limit of less than 10 μU/well of PEPCK activity in a
variety of biological samples.
In conclusion, the Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay Kit provides a robust
and easy to use tool for measuring PEPCK activity in biological matrices and purified samples
(BioVision, Inc. Cat. # K359-100).
Summary: BioVision’s PEPCK Activity Assay Kit is a valuable tool for detecting PEPCK activity in biological
samples and identifying potential PEPCK activity modulators for drug discovery. The assay is
simple to perform, sensitive (≤10 μU/sample) and high-throughput adaptable. The assay kit is
commercially available at www.biovision.com (Cat #K359-100).
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.
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