Phospholipid reactivation of the purified plasma membrane ATPase of yeast.
The plasma membrane ATPase of the yeast Schizosaccharomyces pombe solubilized by lysolecithin and purified by centrifugation through a sucrose gradient is essentially inactive. The phospholipid distribution in the sucrose gradient indicates that inactivation of the ATPase may result from the partial delipidation occurring during purification. Taking into account the 100,000 daltons of the ATPase polypeptide, it is concluded that 74 mol of phospholipids are bound per mol of purified ATP monomer. The ATPase so purified is reactivated simply by mixing the enzyme with preformed lipid micelles or vesicles. Lysolecithins reactivate the enzyme at concentrations around the critical micellar concentration. Gel exclusion chromatography indicates that the enzyme binds reversibly to the lysolecithin micelles. On the other hand, lecithins of varying chain length and unsaturation reactivate the enzyme to different extents and with different efficiencies. In addition, from binding studies, it is observed that each saturated lecithin combines equally well with the ATPase. Using other diacylphospholipids no specificity for the polar head group is observed. Moreover, cardiolipin microvesicles is shown to bind all the protein but not to restore the enzyme activity. From lipid-reactivation titration curves. Arrhenius plots, and physical data of the phospholipids, it is concluded that the major parameter which governs the optimal reactivation of ATPase is the ability of the phospholipids to form amphipathic structures (micelles or vesicles) of sufficient fluidity and hydrophobicity. From these results, a coherent description can be provided for the protein-lipid interactions occurring during solubilization, purification, and the lipid reactivation of the yeast plasma membrane ATPase.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008563:Membrane Lipids / Q000502:physiology; D010743:Phospholipids / Q000494:pharmacology* / Q000502:physiology; D012568:Schizosaccharomyces / Q000201:enzymology*; D013329:Structure-Activity Relationship
D008563:Membrane Lipids; D010743:Phospholipids; D000251:Adenosine Triphosphatases