title: A simple and rapid method for the reversible removal of lipids from a membrane-bound enzyme.
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.
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D002498:Centrifugation; D002592:Cetomacrogol; D003902:Detergents; D008563:Membrane Lipids / Q000032:analysis; D008722:Methods; D010743:Phospholipids / Q000032:analysis; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
chemical_list: D003902:Detergents; D008563:Membrane Lipids; D010743:Phospholipids; D002592:Cetomacrogol; D012964:Sodium; D000251:Adenosine Triphosphatases; D011188:Potassium