Covalent inhibitors of P-glycoprotein ATPase activity.

Verapamil-stimulated ATP hydrolysis by Chinese hamster P-glycoprotein in plasma membranes was shown to occur at a site(s) which is conformationally flexible and of relatively low affinity and specificity. Such properties distinguish P-glycoprotein from other transport ATPases. 8-Azido-ATP and 2-azido-ATP were excellent substrates, confirming that both analogs are suitable photoaffinity labels for investigating the catalytic site(s). Inactivation of ATPase activity occurred coincident with covalent incorporation of approximately two 8-azido-ATP/P-glycoprotein, with the incorporated analog distributed equally between N- and C-terminal halves of the molecule. N-Ethylmaleimide potently inactivated in an ATP-protected, dithiothreitol-irreversible manner, with maximal inactivation occurring coincident with incorporation of approximately two N-ethyl-maleimide/P-glycoprotein. The critical catalytic site sulfhydryls were shown to be located equally in N- and C-terminal halves of the molecule. Sulfhydryl-substituted purines also gave substantial inhibition of P-glycoprotein ATPase activity, which was dithiothreitol reversible. The data provide guidelines for beginning investigation of catalytic site architecture by protein chemistry approaches.