Abstract
A simple, rapid and reproducible method for the reversible removal of lipids from a membrane-bound enzyme is described. Essentially, a membrane preparation containing (Na+ + K+)-dependent adenosine triphosphatase was extracted with the non-ionic detergent Lubrol WX in the presence of glycerol, and partial separation of protein from lipid was achieved with the use of only two centrifugations. About 74% of the endogenous phospholipid and 79% of the cholesterol were removed, concomitant with a virtually complete loss of ouabain-sensitive adenosine triphosphatase activity, but with retention of 60-100% of the K+-dependent phosphatase activity. The addition of pure phosphatidylserine re-activated the enzyme to more than 80% of the initial activity, and up to 30% of the protein was recovered. Excess of phosphatidylserine could be washed off the enzyme to give a stable 'reconstituted' preparation. The effects of variation in the experimental conditions were examined, and the results are discussed with respect to the possibility of adapting the method to the study of other lipid-dependent enzymes bound to membranes.
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Selected References
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- Coleman R. Membrane-bound enzymes and membrane ultrastructure. Biochim Biophys Acta. 1973 Apr 3;300(1):1–30. doi: 10.1016/0304-4157(73)90010-5. [DOI] [PubMed] [Google Scholar]
- Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
- Kimelberg H. K. Protein-liposome interactions and their relevance to the structure and function of cell membranes. Mol Cell Biochem. 1976 Feb 25;10(3):171–190. doi: 10.1007/BF01731688. [DOI] [PubMed] [Google Scholar]
- Palatini P., Dabbeni-Sala F., Bruni A. Reactivation of a phospholipid-depleted sodium, potassium-stimulated ATPase. Biochim Biophys Acta. 1972 Nov 2;288(2):413–422. doi: 10.1016/0005-2736(72)90262-3. [DOI] [PubMed] [Google Scholar]
- Towle D. W., Copenhaver J. H., Jr Partial purification of a soluble (Na+ + K+)--dependent ATPase from rabbit kidney. Biochim Biophys Acta. 1970 Mar 17;203(1):124–132. doi: 10.1016/0005-2736(70)90042-8. [DOI] [PubMed] [Google Scholar]
- Walker J. A., Wheeler K. P. Differential effects of temperature on a membrane adenosine triphosphatase and associated phosphatase. Biochem J. 1975 Nov;151(2):439–442. doi: 10.1042/bj1510439. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walker J. A., Wheeler K. P. Polar head-group and acyl side-chain requirements for phospholipid-dependent (Na-+ plus K-+)-ATPase. Biochim Biophys Acta. 1975 Jun 11;394(1):135–144. doi: 10.1016/0005-2736(75)90212-6. [DOI] [PubMed] [Google Scholar]
- Wheeler K. P., Walker J. A., Barker D. M. Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system. Biochem J. 1975 Mar;146(3):713–722. doi: 10.1042/bj1460713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wheeler K. P., Walker J. A. Differential effects of lipid depletion on membrane sodium-plus-potassium ion-dependent adenosine triphosphatase and potassium ion-dependent phosphatase. Biochem J. 1975 Mar;146(3):723–727. doi: 10.1042/bj1460723. [DOI] [PMC free article] [PubMed] [Google Scholar]