Background: Phosphatidate phosphohydrolase enzyme (PAP) catalyzes the transformation of phosphatidic acid to P_i and diacylglycerol. Two different forms of PAP have been reported in rat hepatocytes PAP₁ which participates in the synthesis of phospholipids and triacylglycerols, and PAP₂ which involved in lipid signaling transduction. PAP₂ has two isoforms namely PAP_2a and PAP_2b. In this study the role of essential histidine residues in PAP_2b was assessed.

Material and Methods: The rat liver plasma membrane was solved using n-octyle glucoside and PAP_2b purified during several chromatography steps. The kinetics parameters were assessed based on surface kinetic dilution model. Discontinuous gel electrophoresis (SDS-PAGE) was performed on purified enzyme in order to evaluate its purity and to measure the molecular weight of the enzyme subunit in gel 10%. The number of mol histidine residues modified per mol enzyme was determined based on the formation of N-carbetoxy histidine.

Results: The specific activity of purified enzyme was 7350mU/mg protein. PAP_2b showed only a single band on SDS-PAGE with a mass weight of 33.8 kDa by electrophoresis. The PAP_2b was inactivated by DEPC. The inhibition was competitive with respect to phosphatidate and the maximum 6 moles of histidine residues were modified per mole PAP_2b.

Conclusion: The data have shown that the incubation of PAP_2b by DEPC plus phosphatidate can prevent the inhibitory effect of DEPC on enzyme activity. Our findings also revealed the role of histidine in the active site of PAP_2b .This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate.