Covalent inhibitors of P-glycoprotein ATPase activity

• Published in: The Journal of biological chemistry 1994-03, Vol.269 (12), p.8986-8992
• Main Authors: EL-SHAWI, M. K, URBATSCH, I. L, SENIOR, A. E
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - antagonists & inhibitors; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - pharmacology; Affinity Labels; Analytical, structural and metabolic biochemistry; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides - pharmacology; Biological and medical sciences; Carrier Proteins - antagonists & inhibitors; Cell Membrane - enzymology; CHO Cells; Cricetinae; Ethylmaleimide - pharmacology; Fluorescein-5-isothiocyanate - pharmacology; Fundamental and applied biological sciences. Psychology; Glycoproteins; In Vitro Techniques; Membrane Glycoproteins - antagonists & inhibitors; Proteins; Sodium-Potassium-Exchanging ATPase - metabolism; Substrate Specificity; Sulfhydryl Reagents - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37065-5

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.