Evidence for Histidine Residues on Plasma Membrane Phosphatidate Phosphohydrolase from Rat Liver

Objective(s) Phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to yield Pi and  diacylglycerol. Two different forms of PAP in rat hepatocyte have been reported. PAP۱ is located in cytosolic and microsomal fractions and participates in the synthesis of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine, whereas the other form of phosphatidate phosphohydrolase (PAP۲) is primarily involved in lipid signaling pathways. In rat liver,PAP۲ has two isoforms; one PAP۲a and another PAP۲b. In this study, essentialhistidine residues were investigatedin native form of rat purified PAP۲b withdiethylpyrocarbonate. Materials and Methods PAP۲b purified from rat liver plasma membrane by solubilizing with n-octyle glucoside and several chromatography steps. Gel electrophoresis (SDS-PAGE) performed on purified enzyme in order to evaluate its purity and to measure the molecular weight of the enzyme subunit. The enzyme inactivated with diethylpyrocarbonate (DEPC) and the number of moles of histidine residues modified per mol of enzyme determined. Results The specific activity of purified enzyme was ۷۳۵۰mU/mg protein and it showed only a single band on SDS-PAGE with a MW of about ۳۳.۸ kDa. The PAP۲b inactivated by DEPC. The maximum ۶ moles of histidine residues modified per mole of PAP۲b, when about ۹۰% of enzyme activity is lost with DEPC. Conclusion The data showed that the incubation of PAP۲b by DEPC can inhibit enzyme activity.  Our findings also, revealed the presence of essential histidines in the structure of PAP۲b which involve in its activity. This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate.  

Keywords:

Diethyl pyrocarbonate , Histidine , Phosphatidate phosphohydrolase , Phosphatidic acid