Binding of a solubilized membrane ATPase to phospholipid bilayers

Abstract

Interaction of purified Streptococcus fecalis membrane ATPase with lipid bilayer membranes was investigated using aqueous dispersions of phospholipids. A liposome-ATPase complex was demonstrated by gel filtration analysis. Binding of the solubilized ATPase to liposomes was studied by differential ultracentrifugation and the determination of binding parameters was based on the measurement of ATPase activity in the liposome-ATPase complex and the concentration of free ATPase in equilibrium with the complex. ATPase combined in phosphatidylcholine liposomes was essentially as active as the solubilized enzyme, and the binding of the ATPase to the liposomes was not reduced by increasing the ionic strength. The ratio of phosphatidylcholine to ATPase in the liposome complex was approximately 11 1 (w/w). The intrinsic association constant for the interaction, determined from Scatchard plots was 7.4·10 7±1.4·10 7 M −1. Phosphatidylcholine liposomes containing stearylamine or cardiolipin were prepared to investigate the effects of surface charge on binding. Stearylamine increased the ATPase binding at low ionic strength. In contrast, cardiolipin did not appear to change the binding characteristics. Phosphatidylethanolamine also had little effect on the ATPase binding. Cholesterol incorporated into phosphatidylcholine liposomes lowered ATPase binding. ATPase binding to synthetic dimyristoyl phosphatidylcholine liposomes was lower than with natural phosphatidylcholine liposomes at 4 °C but not significantly different at 30 °C, above the gel-liquid crystalline phase-transition temperature.