COVALENT MODIFICATION OF HUMAN P-GLYCOPROTEIN MUTANTS CONTAINING A SINGLE CYSTEINE IN EITHER NUCLEOTIDE-BINDING FOLD ABOLISHES DRUG-STIMULATED ATPASE ACTIVITY

被引:175
作者
LOO, TW [1 ]
CLARKE, DM [1 ]
机构
[1] UNIV TORONTO,DEPT MED,MED RES COUNCIL GRP MEMBRANE BIOL,TORONTO,ON M5S 1A8,CANADA
关键词
D O I
10.1074/jbc.270.39.22957
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992). To test this postulate as well as determine the contribution of either nucleotide-binding domain to function, a Cys-less mutant was constructed, and then a single cysteine residue was reintroduced back into each nucleotide binding consensus sequence. We then tested the sensitivity of the ATPase activity of each mutant to covalent modification by NEM, It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys-431 and Cys-1074, respectively) with NEM inhibited drug-stimulated ATPase activity of P-glycoprotein. The concentrations of NEM required for half-maximal inactivation of ATPase activity were 7 and 35 mu M for mutants Cys-431 and Cys-1074, respectively. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide-binding domains may need to bind ATP to couple drug binding to ATPase activity.
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页码:22957 / 22961
页数:5
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