cPGA hydrolase assay of DJ-1 in crude cell lysates: Implications for sensing of oxidative stress

Abstract

Cyclic 3-phosphosphoglyceric anhydride (cPGA), a side product of glycolysis, acylates cellular amines and thiols to form amides and thioesters, respectively. Since these acylation reactions are harmful, organisms rely on a protein, known as DJ-1 in humans, to inactivate cPGA. Inactivation of cPGA likely plays a significant role in cytoprotection by DJ-1, but further progress in this direction is hampered by the lack of quantitative assays to measure the cPGA hydrolase activity of DJ-1 in biological samples. Here we report an optimized procedure for preparation of cPGA which is then used as a substrate to quantify enzymatic activity of DJ-1. The end-point assay for cPGA hydrolase uses dilute cell lysates to hydrolyze cPGA for 0.5–3.5 min followed by conversion of the remaining cPGA into thioester for spectrophotometric quantitation. We illustrate the utility of this assay by showing that higher levels of cPGA hydrolase activity result in better protection from acylation by cPGA. Moreover, the decrease of cPGA hydrolase activity due to oxidation of the catalytic cysteine of DJ-1 under oxidative stress and its subsequent recovery can be monitored using the assay. This relatively simple assay allows functional characterization of DJ-1 in biological samples through quantitative assessment of its cPGA hydrolase activity.