title: Purification of a phosphatase which hydrolyzes phosphatidic acid, a key intermediate in glucolipid synthesis in Acholeplasma laidlawii A membranes.
abstract: A phosphatidic acid phosphatase (PAP; EC 3.1.3.4.), dephosphorylating phosphatidic acid (PA) to diacylglycerol (DAG), was identified and purified from the plasma membrane of Acholeplasma laidlawii A. After four purification steps, including membrane preparation, Tween 20 solubilization, preparative gel electrophoresis and electro-elution, PAP was purified about 400 times to near homogeneity. The molecular weight of PAP was according to SDS-polyacrylamide gel electrophoresis approximately 25 kDa and the enzyme was a stable and integral membrane protein. It is proposed to catalyze the first enzymatic step in the important glucolipid pathway of A. laidlawii. No essential cofactors or activator lipids were found. However, some divalent cations and phosphate analogues were potent inhibitors. Beside the in vivo substrate (PA), PAP was found to dephosphorylate p-nitrophenylphosphate. This less stringent specificity makes alternative in vivo functions for PAP plausible, the importance which is discussed.
authors.affiliation: Department of Biochemistry, Umeå University, Sweden. sbg@chem.umu.se
authors.forename: S
authors.initials: S
authors.lastname: Berg
authors.forename: A
authors.initials: A
authors.lastname: Wieslander
mesh_terms: D000128:Acholeplasma laidlawii / Q000201:enzymology* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism; D006017:Glycolipids / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D010711:Phosphatidate Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004075:Diglycerides; D006017:Glycolipids; D010712:Phosphatidic Acids; D010711:Phosphatidate Phosphatase; D008274:Magnesium