Phosphatidylcholine makes specific activity of the purified Ca(2+)-ATPase from plasma membranes independent of enzyme concentration.
Ca(2+)-ATPase of plasma membranes (PMCA) was isolated from either human or pig red cells by calmodulin-affinity chromatography and supplemented with phosphatidylcholine (PC). The specific activity of the purified PMCA diluted in media with detergent (C(12)E(10)) was very low, and increased with the concentration of the enzyme along a curve that reached the maximum at 8 microg/ml with K(0.5)=1.2-2.5 microg/ml. Such behavior has been described and attributed to self-association of the enzyme (D. Kosk-Kosicka and T. Bzdega, J. Biol. Chem. 263 (1988) 18184-18189). After heat-inactivation, the PMCA was as effective an activator as the intact enzyme, increasing, to the maximum, the specific activity of diluted enzyme with K(0. 5)=2.2 microg/ml. The inactivated PMCA failed to increase the activity of concentrated enzyme, suggesting that activation did not depend on interaction of intact with denatured enzyme molecules. When enough PC was added to the reaction medium to make its final concentration 16-33 microg/ml, the specific activity of the PMCA was maximum and independent of enzyme concentration. Under these conditions, activation by calmodulin lowered to 10%. As a function of the concentration of pure PC, maximum specific activity was reached along a curve with K(0.5)=4 microg/ml. This curve was identical to that of activation at increasing enzyme concentration, suggesting that, in the latter case, activation could have depended on PC contributed to the assay medium by the enzyme. The results show that PC made the purified PMCA solubilized in detergent reach maximum activity at any concentration of the enzyme.
Bredeston
L M
LM
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Junín 956, 1113, Buenos Aires, Argentina.
Rega
A F
AF
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D003902:Detergents; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010713:Phosphatidylcholines / Q000494:pharmacology*; D011489:Protein Denaturation; D012995:Solubility; D013552:Swine
D002147:Calmodulin; D003902:Detergents; D010713:Phosphatidylcholines; D000252:Calcium-Transporting ATPases