期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 8, 页码 5253-5256出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.8.5253
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资金
- NCI NIH HHS [R01 CA80900] Funding Source: Medline
Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s), To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol, Cross-linking was observed in 12 of 125 mutants, The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6, In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate, These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide, Based on these results, we propose a model for the arrangement of the TM segments.
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