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 conformat ionally flexible and of relatively low affinity and specificity. Such properties distinguish P glycoprotein from other transport ATPases. 8- Azido-ATP and a-azido-ATP were excellent substrates, confirming that b oth analogs are suitable photoaffinity labels for investigating the ca talytic site(s). Inactivation of ATPase activity occurred coincident w ith covalent incorporation of approximately two 8-azido-ATP/P-glycopro tein, with the incorporated analog distributed equally between N- and C-terminal halves of the molecule. N-Ethylmaleimide potently inactivat ed in an ATP protected, dithiothreitol-irreversible manner, with maxim al inactivation occurring coincident with incorporation of approximate ly two N-ethylmaleimide/P-glycoprotein. The critical catalytic site su lfhydryls 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 dithiothreito l reversible. The data provide guidelines for beginning investigation of catalytic site architecture by protein chemistry approaches.