期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 278, 期 25, 页码 23171-23179出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M301957200
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资金
- NIGMS NIH HHS [GM50156] Funding Source: Medline
Kinetics of inhibition of ATPase activity of pure mouse Mdr3 P-glycoprotein upon incubation with MgADP and vanadate were studied along with the trapping of [C-14] ADP in presence of vanadate. The presence of verapamil strongly magnified both effects. Inhibition of ATPase was also increased by several other drugs known to bind to drug-binding sites. Inhibition by ADP-vanadate was slow and depended cooperatively on nucleotide binding. Stoichiometry of [C-14] ADP trapping by vanadate was 1 mol/mol P-glycoprotein at full inhibition. Catalytic site mutants prevented [C-14] ADP trapping, whereas interdomain signal communication mutants reduced it in approximate correlation with their effects upon drug stimulation of ATPase. In explanation of the results, we propose that a closed conformation involving dimerization and interdigitation of the two nucleotide-binding domains is necessary to allow inhibition by ADP-vanadate. The results suggest that such a conformation occurs naturally during ATP hydrolysis. It is proposed that in order for the catalytic transition state to form, the two nucleotide-binding domains dimerize to form an integrated single entity containing two bound ATP with just one of the two ATP being hydrolyzed per dimerization event.
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