Covalent inhibitors of P-glycoprotein ATPase activity.

M.K Al-Shawi,I.L Urbatsch,A.E Senior

doi:10.1016/s0021-9258(17)37065-5

M.K Al-Shawi, I.L Urbatsch + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)37065-5

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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.

Highlights

Verapamil-stimulatedATP hydrolysisby Chinese ham- It has been clearly established that, in multidrug-resistant ster P-glycoproteinin plasma membranes was shownto cell lines, drug efflux is an energy-dependent process that reoccur at a site(s) which is conformationally flexible and quires ATP (Bradley et al, 1988; Groset al., 1992).P-glycoproof relativelylow affinity and specificity
InhibiCtoorvsalent of P-glycoprotein inactivating the ATPase activity of P-glycoprotein in plasma membranes, and we report studies of the compounds 8-azidoATP, 2-azido-ATP, and fluorescein isothiocyanate as substrates or inactivators
Because 8-azido-ATP and 2-azido-ATPusu- glycoprotein that werelabeled to the extent of 0.6 and 1.4 ally adopt predominantly thesyn- and anti-conformations, re- moVmol by [a-32P]8-azido-ATP,the distributionof radioactivity spectively, in solution

Summary

EXPERIMENTAL PROCEDURES

Purified plasma membrane preparations were made from CRlRl2 cells or the parent AUXBl cellsas described previously The P-glycoprotein contenoft plasma membranes was initially quantitated by laser densitometry of Coomassie Blue-stained SDS gels a s described in Al-Shawi and Senior (1993)T.wo further procedures were used to verify the accuracy of this analysis. Covalent Labeling of P-glycoprotein in Plasma Membranes varying amountsof purified plasma membranes, quantitated the N-Ethylmaleimide-Plasmamembraneswerepre-equilibratedin total amountof P-glycoprotein in the samplebsy laser densitometryas buffer containing 0.25M sucrose, 0.1mM EGTA, 40 mM Tris-C1, pH 7.4, described above. The molecular sizoef P-glycoprotein polypeptide usefdor stoichiometry calculations was 140 kDa. It is pertinent to note that plasma membranes from the parent AUXceBlls showed only minor Coomassie Blue staining in SDS gels at the position of P-glycoprotein (Al-Shawi and Senior, 1993), such that errors introduced into stoichiometry calculations by reaction of labeling reagents with proteins other thaPn-glycoprotein are negligible. We showed previously that low concentrations of dimethyl sulfoxide (as added here with the FITC) do notinhibit P-glycoprotein ATPase activity (Al-Shawi and Senior, 1993)

Routine Procedures

X 104 ND ND

C R l R 1 3

Findings

DISCUSSION