Introduction: PHOSPHATIDATE PHOSPHOHYDROLASE (PAP) catalyzes the dephosphorylation of phosphatidic acid to yield Pi and diacylglycerol. Two forms of PAP in rat hepatocyte have been reported. A cytosolic form (PAP1) that is responsible for glycerolipid metabolism and requires Mg2for its activity. Another form (PAP2) is primarily involved in lipid signaling pathways which dose not need Mg2+. It has two isoforms; PAP 2a and PAP2b. Little information is known about enzymological characterization of PAP2 especially its substrate. We investigated the enzyme behavior against Mg2+ structure- breaking agents (urea and guanidine HCl) with respect to its substrate. Materials & Methods: PAP2b was purified from rat hepatocyte membrane using a multi – stage chromatography. The enzyme activity was determined against La (lamellar) and HII (Hexagonal) forms of substrate in the presence of triton X-100. The effect of Mg2+ concentration and urea and guanidine HCl (structure- breaking agents) were examined on La to HII phase transition and enzyme activity. Results: PAP2b consumes La form of PHOSPHATIDATE. Cations such as Mg2+ result in HII from La phase of substrate and therefore reduce enzyme activity. Urea and guanidine HCl increase enzyme activity due to prevention of HII formation. Both can also reduce the effect of cations on La to HII transition of PHOSPHATIDATE.Conclusion: Since PAP2b consumes La form of substrate parameters can induce La to HII phase transition which result in low enzyme activity. In contrast, factors such as urea and guanidine HCl increase activity due to prevention of La to HII phase transition . As Mg2+ stimulates HII formation and the enzyme needs La form substrate, it can be concluded that, Not only does the enzyme need no Mg2+,but it is controlled by it too.

PHOSPHATIDATE PHOSPHOHYDROLASE (PAP2b, fraction b) was purified from the plasma membrane of rat liver cells. The Km for the surface concentration of phosphatidic acid was 0.43 mol%. The subunit of the enzyme had an M.W. of 33.8 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native enzyme shows a molecular weight of 182 kDa in a gel filtration column packed with Sephacryl S300 in the presence of Triton X-100. The pH optima obtained for PAP2b were 5.5 and 7 in imidazole and Tris- HCl buffers, respectively. The membrane homogenate enzyme (PAP2) consumed the lamellar (La) phase of PHOSPHATIDATE and was activated (approximately 3-fold) by Lubrol PX, CTAB and Tween 80 and inhibited by Zn2+ and Mn2+. The inhibition was concentration dependent. These cations affected PAP2b activity through the phase transition of PHOSPHATIDATE from lamellar (La) to inverted hexagonal (HII) form. Guanidine hydrochloride and urea increased PAP2 activity (2-fold) up to 20mM concentrations by stabilizing the La phase. Optimum activity of purified PAP2b was obtained at 3% trehalose and 7% sucrose. The data suggested that the stability of the La form of PHOSPHATIDATE by detergent micelles may take place through surface dilution processes.

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.

Introduction: PHOSPHOHYDROLASE PHOSPHATIDATE enzyme (PAP) is of two pap 1 and pap2 forms, pap 1 has a role in three acil glyserol as well as other compound phospholypidates while pap2 with two pap2a and PAP2b takes its role in message transmission phenomenon. There is little information about pap2 activity regulation in hand. so, this study has investigated the role played by lysine amino acid in PAP2b enzyme catalytic activity.Methods: In this study, carried out in 2003, PAP2b was extracted from rat liver using a multi-stage chromatography, then it was electrophorized under enzyme unit, to determine the number and weight. the enzyme inactivation and spectophotometric studies of PAP2b was done using pyridoxal 5 phosphate, then, the results were analysed under linear regression and lineweaver- burk diagram.Conclusion: The finds prove the essential lysine presence in the enzyme activity area. It was also concluded that the balanced number of lysine mole was 6 accound for each enzyme mole.  

Background: PHOSPHATIDATE PHOSPHOHYDROLASE (PAP) catalyzes the dephosphorylation of phosphatidic acid to yield Pi and diacylglycerol. Two different forms of PAP have been reported in rat hepatocyte: PAP1 that participates in the synthesis of phospholipids and triacylglycerols and PAP2 which is involved in lipid signaling pathways. Two isoforms of PAP2 are PAP2a and PAP2b. This study examines the effect of detergents such as Tween 80 lubrol, PX and CTAB as well as stabilizing factors including trehalose, sucrose, and albumin on the stability and activity of PAP2b. Methods: 14 Wistar rats weighing between 200-250 grams were used in the study. PAP2b was purified from liver plasma membrane by solubilizing with n-octyle glucoside and through several chromatography stages. Gel electrophoresis (SDSPAGE) was performed in 10%gel slab in order to determine the purity level and to measure the molecular weight and number of the enzyme subunit. The effect of trehalose, sucrose, and albumin was examined on the stimulation of enzyme activity in different concentrations. Results: The specific activity of purified enzyme was 7350 mU/mg protein. The purified enzyme showed a single band on SDS-PAGE with a MW of about 33.8 kDa. The enzyme was approximately activated 3times by lubrol PX and Tween 80 both at 3 mM. The activation by CTAB occurred at 1Mm.Trehalose, sucrose and albumin had the most stability effect on PAP2b in concentration of 3, 7 and 10 percent respectively. Conclusions: Tween 80, lubrol PX, and CTAB have the ability to activate PAP2b. In case a nondetergent agent is required to stabalize PAP2b trehalose is preferred to sucrose and albumin. The lubrol PX has a higher potential stimulatory effect to activate PAP2b. compared to other ionic and nonionic detergents.