The effects of lipids and detergents on ATPase-active P-glycoprotein.

We previously isolated and characterized a partially purified preparation of ATPase-active P-glycoprotein, the multidrug transporter (Doige, C.A., Yu, X. and Sharom, F.J. (1992) Biochim. Biophys. Acta 1109, 149-160). The effect of various detergents and membrane phospholipids on the ATPase activity of P-glycoprotein has now been investigated. P-Glycoprotein ATPase activity was most stable in CHAPS, with over 50% of the activity retained at a concentration of 8 mM. Octyl glucoside in the low mM range also supported the ATPase, while deoxycholate destroyed all activity at 1 mM. Digitonin and SDS inhibited ATPase activity at very low concentrations. Triton X-100 at 2-10 microM stimulated the ATPase almost 2-fold, while higher levels inhibited activity. Although P-glycoprotein ATPase was sensitive to thermal inactivation, full activity was preserved in the presence of asolectin, but not phosphatidylcholine species. Further studies revealed that asolectin, both saturated and unsaturated phosphatidylethanolamines, and phosphatidylserine, were best able to maintain ATPase activity at 23 degrees C. Saturated phosphatidylethanolamine species activated P-glycoprotein ATPase up to 40% at 23 degrees C, and 80% at 4 degrees C. Following detergent delipidation, various lipids were able to restore P-glycoprotein ATPase activity. Unsaturated phosphatidylcholine and phosphatidylserine were most effective, while saturated species were not able to restore catalytic activity. These results indicate that membrane lipids are necessary for catalytic activity of the ATPase domains of P-glycoprotein. P-Glycoprotein has well-defined lipid preferences, with saturated phosphatidylethanolamines both activating the ATPase and providing protection from thermal inactivation, while fluid lipid mixtures are able to restore activity following delipidation.