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. PAP1 is located in cytosolic and microsomal fractions and participates in the synthesis of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine, whereas the other form of PHOSPHATIDATE PHOSPHOHYDROLASE (PAP2) is primarily involved in lipid signaling pathways. In rat liver, PAP2 has two isoforms; one PAP2a and another PAP2b. In this study, essential histidine residues were investigated in native form of rat purified PAP2b with diethylpyrocarbonate.Materials and Methods: PAP2b 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 7350mU/mg protein and it showed only a single band on SDSPAGE with a MW of about 33.8 kDa. The PAP2b inactivated by DEPC. The maximum 6 moles of histidine residues modified per mole of PAP2b, when about 90% of enzyme activity is lost with DEPC. Conclusion The data showed that the incubation of PAP2b by DEPC can inhibit enzyme activity. Our findings also, revealed the presence of essential histidines in the structure of PAP2b which involve in its activity. This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate.

PHOSPHATIDATE PHOSPHOHYDROLASE (PAP) from cytosolic fraction of rat liver was purified to homogeneity having specific activity of 5.14 U/mg proteins. An activity staining procedure was developed to determine molecular weight of the enzyme on polyacrylamide gel electrophoresis using Ferguson plot. Molecular Weight (M.W.) of the active PAP was 298 KDa. SDS-PAGE analysis showed a M.W. of 47 KDa for PAP subunits. Active multimer of the enzyme, therefore, was calculated to be hexamer. Gel filtration on Sephadex G-100 column showed a M.W. of 850 KDa for PAP due to the protein aggregation on the matrix. The purified enzyme was inhibited by divalent cations such as Fe2+, Cu2+ and Ca2+ but requires Mg 2+ for its activity. The activity loss of PAP inhibited by cations was restored by Mg2+ on polyacrylamide gel. The data suggest that the active form of cytosolic PAP is a hexamer of identical subunits and that charge density plays an important role in enzyme-substrate interaction. Magnesium ion is probably the only divalent cation capable of generating proper enzyme-metal-substrate complex necessary for the catalytic activity.

Variations in PHOSPHATIDATE PHOSPHOHYDROLASE (PAP) activity and triacylglycerol concentration were measured in pregnant and hormone-treated non-pregnant female rats. PAP activity in adipose tissue was elevated by 61% during pregnancy. The increase in the enzyme activity was paralleled with a rise in serum triacylglycerol concentration (44%). Estradiol injecting into non-pregnant rats increased PAP activity of both adipose tissue (19.8%) and the liver (26%). Progesterone also elevated the enzyme activities of adipose tissue and liver by 10% and 55%, respectively. Both hormones increased serum triacylglycerol concentration (20-29%). The data demonstrated that hypertriglyceridemia observed during pregnancy was mediated through the hormonal effects on PAP activity, a key enzyme in glycerolipid metabolism.

Soluble PHOSPHATIDATE PHOSPHOHYDROLASE (PAP) from rat liver was purified and inactivated by pyridoxal 5-phosphate (P.L.P). The inhibition was noncompetitive with respect to PHOSPHATIDATE. The loss of the enzyme activity was pseudo-first order and time-dependent. With a 45-fold molar excess of PLP the activity loss was completed in 32 minutes. Specteral charasteristics of PLP-enzyme complex indicated the formation of a Schiffs base between PLP and lysine residue(s) of the enzyme. Kinetic studies demonstrated that one PLP molecule was bound per active unit of the enzyme with a Ki of 0.26 mM. The role of the lysine residue in the catalytic activity of PAP is discussed.

PHOSPHATIDATE PHOSPHOHYDROLASE from rat liver soluble fraction was partially purified. The specific activity was 265 mU/mg protein. The method involved ammonium sulfate fractionation and chromatography on Sephadex G-75, DEAE-Sephadex and CMSephadex. The enzyme was inactivated with diethylpyrocarbonate. Spectral and kinetic data suggested that the inactivation resulted from the modification of histidine residues which participate in binding and/or catalytic activity of the enzyme.

The mechanism by which bi-and trivalent cations affect human liver PHOSPHATIDATE PHOSPHOHYDROLASE (PAP) activity was investigated. Bivalent cations up to 1 mM increased PAP activity whereas at higher concentrations the activity of the enzyme decreased. The stimulatory concentration for trivalent cations such as Al3+ and Cr3+, however, was much lower being 2 m M and 1 m M, respectively. All cations affecting PAP activity were also able to induce phase transition of PHOSPHATIDATE from lamellar (La) to inverted hexagonal (HII) form. The rate of La-HII transition was different for each cation. At 100 mM concentration of Mg2+ only 26% of the original PHOSPHATIDATE remained in La form and for other cations tested ranged from 14.5% to 76%. The phase transition was blocked by EDTA. Magnesium from 0.8 to 1.5 mM concentration raised PAP activity (3-fold) with La form of substrate but not with the HII phase. Monovalent cations such as Na+ and K+ neither affected enzyme activity nor substrate configuration. These data suggest that cation-induced PAP activation is not as a result of cation-protein interaction, but is due to formation of a suitable substrate configuration for the enzyme catalysis during PHOSPHATIDATE phase transition. It appears that the real substrate configuration for PAP activity is situated between La and HII phases.