Characterization of a phosphatidic acid phosphatase from rat brain cell membranes.
We have characterized a phosphatidic acid phosphatase (PAP, EC 3.1.3.4) that is associated with cell membranes from rat brain using [32P]phosphatidic acid as substrate in a simple assay. The enzyme could be activated by Triton X-100, cholic acid and Chaps and inhibited by Lubrol PX and sodium dodecyl sulfate. The optimal pH was between 6.0 and 7.0 Mg2+ was not essential for enzyme activity. The enzyme activity was decreased by about 50% by Ca2+ at concentrations of 0.1 to 1 mmol/l. Zn2+ inhibited the enzyme by 50% at concentrations of about 10 mumol/l in the absence of, and 100 nmol/l in the presence (3 mmol/l) of, Triton X-100. NaF decreased the activity by about 50% at concentrations between 0.3 and 1 mmol/l when Triton X-100 was added, but did not inhibit the enzyme if the detergent was not present. N-Ethylmaleimide (NEM) did not affect the enzyme. In the absence of Triton X-100, propranolol and metoprolol enhanced the PAP activity. In the presence of 3 mmol/l Triton X-100, the enzyme was inhibited by about 50% by propranolol at a concentration of 10 mmol/l, whereas metoprolol caused only a slight inhibition of PAP. The Km for phosphatidic acid was 150 mumol/l and was changed to 20 mumol/l by 3 mmol/l Triton X-100 without the Vmax being changed. Enzyme activity could be solubilized by 1-5% (w/v) Triton X-100. Gel filtration chromatography showed a M(r) of 320,000. This membrane-associated PAP from neuronal tissue probably belongs among the NEM-insensitive forms of PAP enzymes which have been proposed to play a role in transmembrane signal transduction via phospholipase D.
Höer
A
A
Institut für Pharmakologie, Freien Universität Berlin, Germany.
Oberdisse
E
E
D000319:Adrenergic beta-Antagonists; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002412:Cations; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D008790:Metoprolol / Q000494:pharmacology; D017830:Octoxynol; D010711:Phosphatidate Phosphatase / Q000737:chemistry*; D011433:Propranolol / Q000494:pharmacology; D051381:Rats
D000319:Adrenergic beta-Antagonists; D002412:Cations; D003902:Detergents; D017830:Octoxynol; D011433:Propranolol; D010711:Phosphatidate Phosphatase; D008790:Metoprolol