期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 26, 页码 19435-19438出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.C000222200
关键词
-
资金
- NCI NIH HHS [R01 CA80900] Funding Source: Medline
P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide-binding domains (NBDs), Disulfide cross-linking analysis was done to determine if the two NBDs are close to each other. Residues within or close to the Walker A (GNSGCGKS in NDB1 and GSSGCGKS in NBD2) sequences for nucleotide binding were replaced with cysteine, and the mutant P-gps were subjected to oxidative cross-linking. Cross-linking was detected in two mutants, G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2), because the crosslinked proteins migrated slower in SDS gels. Mutants G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2) retained 10% and 82%, respectively, of the drug-stimulated ATPase activity relative to that of Cysless P-gp. The cross-linking properties of the more active mutant L439C(NBD1)/Cys-1074(NBD2) were then studied. Cross-linking was reversed by addition of dithiothreitol and could be prevented by pretreatment of the mutant with N-ethylmaleimide. Cross-linking was also inhibited by MgATP, but not by the verapamil. Oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) resulted in almost complete inhibition of drug-stimulated ATPase activity. More than 60% of the drug-stimulated ATPase activity, however, was recovered after treatment with dithiothreitol, The results indicate that the two predicted nucleotide-binding sites are close to each other and that cross-linking inhibits ATP hydrolysis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据