| Abstrakt: |
Reconstitution of purified rabbit kidney Na,K-ATPase in phosphatidylcholine/phosphatidic acid liposomes resulted in the absence of ATP in a time-, temperature- and protein-dependent formation of inorganic phosphate. This formation of inorganic phosphate could be attributed to a phosphatidate phosphohydrolase activity present in the Na,K-ATPase preparation. A close interaction of the enzyme with the substrate phosphatidic acid was important, since no or little Pi production was observed under any of the following conditions: without reconstitution, after reconstitution in the absence of phosphatidic acid, with low concentrations of detergent or at low lipid/protein ratios. The hydrolysis of phosphatidic acid was not influenced by the Na,K-ATPase inhibitor ouabain but was completely inhibited by the P-type ATPase inhibitor vanadate. Besides Pi diacylglycerol was also formed, confirming that a phosphatidate hydrolase activity was involved. Since the phosphatidate phosphohydrolase activity was rather heat- and N-ethylmaleimide-insensitive, we conclude that the phosphatidic acid hydrolysis was not due to Na,K-ATPase itself but to a membrane-bound phosphatidate phosphohydrolase, present as an impurity in the purified rabbit kidney Na,K-ATPase preparations. |
