Evidence for histidine residues on plasma membrane phosphatidate phosphohydrolase from rat liver

Phosphatidate phosphohydrolase [PAP] catalyzes the dephosphorylation of phosphatidic acid to yield P[i], and diacylglycerol. Two different forms of PAP in rat hepatocyte have been reported. PAP[1] is located in cytosolic and microsomal fractions and participates in the synthesis of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine, whereas the other form of phosphatidate phosphohydrolase [PAP[2]] is primarily involved in lipid signaling pathways. In rat liver, PAP2 has two isoforms; one PAP[2a] and another PAP2b- In this study, essential histidine residues were investigated in native form of rat purified PAP[2b] with diethylpyrocarbonate. PAP[2b] 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. The specific activity of purified enzyme was 7350mU/mg protein and it showed only a single band on SDS-PAGE with a MW of about 33.8 kDa. The PAP[2b] inactivated by DEPC. The maximum 6 moles of histidine residues modified per mole of PAP[2b], when about 90% of enzyme activity is lost with DEPC. The data showed that the incubation of PAP[2b] by DEPC can inhibit enzyme activity. Our findings also, revealed the presence of essential histidines in the structure of PAP[2b] which involve in its activity. This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate

Inactivation of a beta-glucosidase from Arthrobotrys conoides by diethyl pyrocarbonate: evidence of histidine at the active site.

Modification of A. conoides beta-glucosidase by diethylpyrocarbonate caused rapid inactivation of the enzyme. The kinetic analyses showed that the inactivation by diethylpyrocarbonate resulted from the modification of an average of one histidine residue per mole of enzyme. The modified enzyme showed an increase in absorbance at 240 nm. Sulphydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. The substrate offered significant protection against diethylpyrocarbonates modification. The results indicate that diethylpyrocarbonate was interacting with the enzyme at or near the active site.

Inactivation of leucine aminotransferase with diethylpyrocarbonate and rose bengal: evidence for an active site histidine residue.

Modification of leucine aminotransferase by diethylpyrocarbonate or rose bengal-sensitized photo-oxidation caused rapid inactivation of the enzyme. The inactivation of leucine aminotransferase depended on the concentration of the reagent, the time of incubation and exhibited pseudo-first order kinetics. Rose bengal-sensitized photo-oxidation was maximum at pH 6.5 and 9. Substrates leucine and alpha-ketoglutarate protected the enzyme against inactivation by these reagents, thus suggesting participation of histidine residue at the substrate binding site.