Vanadate-sensitive phosphatidate phosphohydrolase activity in a purified rabbit kidney Na,K-ATPase preparation.
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.
Swarts
H G
HG
Department of Biochemistry, University of Nijmegen, Netherlands.
Moes
M
M
Schuurmans Stekhoven
F M
FM
De Pont
J J
JJ
D000818:Animals; D002855:Chromatography, Thin Layer; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014638:Vanadates / Q000494:pharmacology*
D008081:Liposomes; D010710:Phosphates; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011510:Proteolipids; C020485:proteoliposomes; D014638:Vanadates; D010042:Ouabain; D010711:Phosphatidate Phosphatase; D000254:Sodium-Potassium-Exchanging ATPase