Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Mr 170,000 to 180,000 membrane glycoprotein associated with multidrug resistance (P-glycoprotein) is involved in drug transport mechanisms across the plasma membrane of multidrug-resistant cells. We have recently reported the purification of P-glycoprotein. The purified P-glycoprotein was found to have an ATPase activity, which might be coupled with the active efflux of anticancer drugs. In the present study, we have further studied the properties of the P-glycoprotein ATPase activity by an immobilized enzyme assay procedure using a P-glycoprotein-antibody-Protein A-Sepharose complex. GTP was also hydrolyzed by the P-glycoprotein, although less efficiently than ATP. The ATPase activity of P-glycoprotein had an optimal pH range around neutrality (pH 6.5-7.4). The detergent concentration of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate used for protein solubilization was essential for enzyme recovery. Maximum activity was obtained when 0.1-0.2% 3-[(3-cholamidopropyl)dimethyl-ammonio]-propane sulfonate was used, while higher concentrations markedly inhibited the ATPase activity. The ATPase activity was dependent on Mg2+; maximum activity was obtained at 2-10 mM. Manganese and cobalt could substitute for magnesium as ionic cofactors. Divalent cations such as Ca2+, Zn2+, Ni2+, Cd2+, and Cu2+ inhibited the Mg2+-catalyzed ATP hydrolysis. N-Ethylmaleimide and vanadate inhibited the ATPase activity, while sodium azide or ouabain had no effect. Anticancer agents such as vincristine and Adriamycin did not affect the enzyme activity. In contrast, verapamil and trifluoperazine, agents which inhibit active drug efflux and restore drug sensitivity in resistant cells, caused an increase in the P-glycoprotein ATPase activity suggesting that P-glycoprotein might be the target molecule of these agents.
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PMID:Characterization of the ATPase activity of the Mr 170,000 to 180,000 membrane glycoprotein (P-glycoprotein) associated with multidrug resistance in K562/ADM cells. 290 Jun 77

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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PMID:ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells. 762 49

Verapamil-stimulated ATP hydrolysis by Chinese hamster P-glycoprotein in plasma membranes was shown to occur at a site(s) which is conformationally flexible and of relatively low affinity and specificity. Such properties distinguish P-glycoprotein from other transport ATPases. 8-Azido-ATP and 2-azido-ATP were excellent substrates, confirming that both analogs are suitable photoaffinity labels for investigating the catalytic site(s). Inactivation of ATPase activity occurred coincident with covalent incorporation of approximately two 8-azido-ATP/P-glycoprotein, with the incorporated analog distributed equally between N- and C-terminal halves of the molecule. N-Ethylmaleimide potently inactivated in an ATP-protected, dithiothreitol-irreversible manner, with maximal inactivation occurring coincident with incorporation of approximately two N-ethyl-maleimide/P-glycoprotein. The critical catalytic site sulfhydryls were shown to be located equally in N- and C-terminal halves of the molecule. Sulfhydryl-substituted purines also gave substantial inhibition of P-glycoprotein ATPase activity, which was dithiothreitol reversible. The data provide guidelines for beginning investigation of catalytic site architecture by protein chemistry approaches.
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PMID:Covalent inhibitors of P-glycoprotein ATPase activity. 790 96

A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose. The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors. There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection. Vanadate was also an effective inhibitor. Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp. The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.
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PMID:Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein. 791 80

A multidrug-resistant Chinese hamster ovary cell line (CR1R12) was obtained which constitutively expresses P-glycoprotein, up to 32% by weight of plasma membrane protein. CR1R12 plasma membranes had high, drug-activated ATPase activity referable to P-glycoprotein. The specific ATPase activity in the presence of verapamil was calculated to be approximately 9 mumol/min/mg (identical to 21 s-1) at 37 degrees C, pH 7.4. KM ATP was 1.4 mM, and ADP and 5'-adenylyl imidodiphosphate were competitive inhibitors with Ki values 0.35 and 0.44 mM, respectively. 2'-dATP was a good substrate, GTP and ITP were real but poor substrates, and ADP and AMP were not hydrolyzed. Optimal pH for ATP hydrolysis was 7.3. MgATP was the preferred substrate, and CaATP was hydrolyzed very weakly. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) covalently labeled the P-glycoprotein, and incorporation of 1.1 mol of NBD-Cl/mol of P-glycoprotein gave 100% inactivation. ATP protected against NBD-Cl inactivation. N-Ethylmaleimide was a potent inhibitor in the absence of ATP, and in its presence significant protection from inhibition could be achieved. Vanadate and fluoroaluminate were also strong inhibitors. The plasma membranes from CR1R12 cells should provide material for purification and reconstitution of P-glycoprotein and for screening of potential "multidrug-reversal" reagents by enzymic assay.
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PMID:Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein. 809 47