title: Covalent inhibitors of P-glycoprotein ATPase activity.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Rochester Medical Center, New York 14642.
authors.forename: M K
authors.initials: MK
authors.lastname: al-Shawi
authors.forename: I L
authors.initials: IL
authors.lastname: Urbatsch
authors.forename: A E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D016466:CHO Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D005033:Ethylmaleimide / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D013439:Sulfhydryl Reagents; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D005033:Ethylmaleimide