A High‐Throughput Assay to Measure Phosphoenolpyruvate Carboxykinase

Abstract

Phosphoenolpyruvate Carboxykinase (PEPCK EC 4.1.1.32) catalyzes the conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP) in the presence of GTP. In humans, two isoforms of PEPCK are found: cytosolic form (PEPCK‐C, also called PCK1) and mitochondrial form (PEPCK‐M, also called PCK2). PEPCK‐C (PCK1) is a rate‐controlling enzyme in gluconeogenesis. Recent studies found abnormal concentrations of PEPCK in diabetic mice. Overexpression of PEPCK attenuates insulin signaling and decreases hepatic insulin sensitivity in transgenic mice. Our high throughput assay for Phosphoenolpyruvate Carboxykinase activity provides a valuable tool for both mechanistic and therapeutic studies of diabetes as well as avenue for targeting PEPCK activity.The Phosphoenolpyruvate Carboxykinase activity assay is developed by using a set of specific enzymes that convert Phosphoenolpyruvate (PEP) and carbonate into a series of intermediates and hydrogen peroxide, which in turn, reacts with a probe generating a colorimetric signal (OD570 nm). The color intensity is directly proportional to the amount of active Phosphoenolpyruvate Carboxykinase. This assay is simple, sensitive, high‐throughput adaptable and can detect less than 10 μU of Phosphoenolpyruvate Carboxykinase activity per sample.In conclusion, a Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay kit is developed. The kit provides easy and robust tool for measuring PEPCK activity in biological and purified samples (BioVision, Inc. Cat. # K359‐100).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.