Characterization of a phosphatidic acid phosphatase from rat brain cell membranes |
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Authors: | Ariane Höer Eckard Oberdisse |
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Institution: | (1) Institut für Pharmakologie der Freien Universität Berlin, Thielallee 69-73, D-14195 Berlin, Germany |
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Abstract: | 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/1. Zn2+ inhibited the enzyme by 50% at concentrations of about 10 mol/l in the absence of, and 100 nmol/1 in the presence (3 mmol/1) 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/1 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 K
m for phosphatidic acid was 150 mol/1 and was changed to 20 mol/1 by 3 mmol/1 Triton X-100 without the V
max being changed. Enzyme activity could be solubilized by 1–5% (w/v) Triton X-100. Gel filtration chromatography showed a M
r of 320000. 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.
Correspondence to: Ariane Hoer at the above address |
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Keywords: | Phosphatidic acid phosphatase Phospholipids Detergents Cationic amphiphilic compounds (\-adrenergic receptor antagonists) |
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