Lipid-protein interactions of reconstituted membrane-associated adenosinetriphosphatases. Use of a gel-filtration procedure to examine phospholipid-activity relationships.
A gel-filtration procedure is described for the reconstitution of partially delipidated membrane adenosinetriphosphatases (Mg2+-ATPase and (Na+ + K+)-ATPase) into liposomes of defined composition. After detergent solubilization of membrane enzyme preparations, reconstitution of these ATPases was achieved by the rapid removal of deoxycholate by Sephadex G-50 chromatography. Proteoliposomes were separated from unincorporated enzyme by chromatography on Sepharose CL-4B. Sedimentation characteristics in sucrose density gradients and electron microscopy confirmed that both Mg2+-ATPase and (Na+ + K+)-ATPase were reconstituted into liposomes of phosphatidylcholine and yielded preparations having high recoveries of enzyme activity by comparison with the control membrane preparations. Reconstitution of (Na+ + K+)-ATPase into synthetic phosphatidylcholines of defined fatty acid composition reveals an inverse relationship between enzyme activity and the chain length of the saturated fatty acids DMPC, DPPC and DSPC. Higher recoveries were obtained when one or more fatty acid chains was unsaturated. Full reactivation occurred with DOPC (18:1/18:1). There was a positive correlation between the specific activity of reconstituted (Na+ + K+)-ATPase and the temperature of the thermal phase transition of the synthetic phosphatidyl cholines studied. This was not seen with Mg2+-ATPase. It is suggested that 'membrane fluidity' influences the catalytic activity of (Na+ + K+)-ATPase but not that of Mg2+-ATPase.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002540:Cerebral Cortex / Q000201:enzymology*; D008081:Liposomes; D008563:Membrane Lipids / Q000502:physiology; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010743:Phospholipids; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D000254:Sodium-Potassium-Exchanging ATPase