Abstract
ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.
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Abbreviations
- AMPPNP:
-
adenyl-5′-yl-imidodiphosphate
- TNP-ATP:
-
2′,3′-O-(2,4,6)-trinitrophenyl-ATP
- BzATP:
-
3′-O-(4-benzoyl)benzoyl-ATP
- ATPγS:
-
adcnosine-5′-O-(3-thiotriphosphate)
- DCCD:
-
dicyclohexylcarbodiimide
- DTT:
-
dithiothreitol
- FITC:
-
fluorescein isothiocyanate
- NEM:
-
N-ethylmaleimide
- NBD-Cl:
-
7-chloro-4-nitrobenzo-2-oxa-1.3-diazole
- TPA+ :
-
tetraphenylarsonium ion: TPP+, tetraphenylphosphonium ion
- Pgp:
-
P-glycoprotein
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Senior, A.E., Al-Shawi, M.K. & Urbatsch, I.L. ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells. J Bioenerg Biomembr 27, 31–36 (1995). https://doi.org/10.1007/BF02110328
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DOI: https://doi.org/10.1007/BF02110328