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)

In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.
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PMID:ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. 135 66

To study the molecular function of the multidrug-resistance gene product P-glycoprotein, we purified and reconstituted it into liposomes. Twelve detergents were examined in an attempt to solubilize and reconstitute the transport activity of K562/ADM membrane proteins containing P-glycoprotein. We found that transport activity was effective reconstituted after solubilization with cholate, glycocholate and taurocholate. Other detergents, such as CHAPS, Triton X-100 and deoxycholate, diminished the transport activity. The K562/ADM membrane was solubilized by 1% glycocholate, and P-glycoprotein was purified by MRK-16 immunoaffinity column chromatography to a homogeneous single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified P-glycoprotein was reconstituted by detergent dialysis into liposomes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The reconstituted P-glycoprotein specifically bound [3H]azidopine and had an ATPase activity that was slightly stimulated when vincristine was added. Furthermore, though its activity was reduced, the reconstituted P-glycoprotein was shown to be an ATP-dependent transporter of vincristine.
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PMID:Reconstitution of purified P-glycoprotein into liposomes. 755 41

P-glycoprotein (P-gp) is thought to function as a drug efflux pump in multidrug resistant (MDR) cells. The functional form of P-gp in its native state is not known. Previous results from radiation target size analysis have suggested that P-gp occurs as dimers in MDR cell plasma membranes [Boscoboinik et al. (1990) Biochim. Biophys. Acta 1027, 225-228]. In this study, we used sucrose gradient velocity sedimentation to determine if P-gp oligomers could be retrieved from detergent extracts of hamster and human MDR cell lines. The proportion of P-gp recovered as higher order oligomers was dependent on the detergents used for solubilization of the cells. When a detergent such as CHAPS was used, 50% or more of the P-gp sedimented as higher order oligomers. In contrast, in the presence of SDS, only monomers were retrieved, but naturally occurring oligomers could be preserved if the cells were treated with a cross-linker prior to detergent solubilization. The oligomers and monomers were both able to bind the photoactive analog of ATP (8-azido[alpha-32P]ATP) or the drug [3H]azidopine in membrane preparations. P-gp is a phosphoprotein, and its phosphorylated state is thought to be important for function. When MDR cells were labeled with [32P]orthophosphate in vivo, we observed that the monomer and dimer were more highly phosphorylated than the larger oligomers, suggesting that these different forms of P-gp may be functionally distinct. The assembly of oligomers appears to occur in an early bisynthetic compartment, and asparagine-linked glycosylation is not required for their formation. Our findings indicate that oligomers of P-gp exist in MDR cells and raise the possibility that the dynamics of oligomer formation and dissociation may be important in the mechanism of action of P-gp.
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PMID:Detection of oligomeric and monomeric forms of P-glycoprotein in multidrug resistant cells. 790 29

We previously isolated and characterized a partially purified preparation of ATPase-active P-glycoprotein, the multidrug transporter (Doige, C.A., Yu, X. and Sharom, F.J. (1992) Biochim. Biophys. Acta 1109, 149-160). The effect of various detergents and membrane phospholipids on the ATPase activity of P-glycoprotein has now been investigated. P-Glycoprotein ATPase activity was most stable in CHAPS, with over 50% of the activity retained at a concentration of 8 mM. Octyl glucoside in the low mM range also supported the ATPase, while deoxycholate destroyed all activity at 1 mM. Digitonin and SDS inhibited ATPase activity at very low concentrations. Triton X-100 at 2-10 microM stimulated the ATPase almost 2-fold, while higher levels inhibited activity. Although P-glycoprotein ATPase was sensitive to thermal inactivation, full activity was preserved in the presence of asolectin, but not phosphatidylcholine species. Further studies revealed that asolectin, both saturated and unsaturated phosphatidylethanolamines, and phosphatidylserine, were best able to maintain ATPase activity at 23 degrees C. Saturated phosphatidylethanolamine species activated P-glycoprotein ATPase up to 40% at 23 degrees C, and 80% at 4 degrees C. Following detergent delipidation, various lipids were able to restore P-glycoprotein ATPase activity. Unsaturated phosphatidylcholine and phosphatidylserine were most effective, while saturated species were not able to restore catalytic activity. These results indicate that membrane lipids are necessary for catalytic activity of the ATPase domains of P-glycoprotein. P-Glycoprotein has well-defined lipid preferences, with saturated phosphatidylethanolamines both activating the ATPase and providing protection from thermal inactivation, while fluid lipid mixtures are able to restore activity following delipidation.
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PMID:The effects of lipids and detergents on ATPase-active P-glycoprotein. 809 61

The use of anticancer drugs in the chemotherapeutic treatment of cancer patients frequently results in the emergence of drug resistant tumors. Selection of tumor cell lines in vitro has led to the identification of several proteins that mediate drug resistance to anticancer drugs. In this study, an immuno-dot blot method was used to isolate a monoclonal antibody (IPM96) which recognized a 40 kDa protein (or P-40) co-expressed with P-glycoprotein and MRP in several multidrug resistant cell lines (MCF-7/Adr, SKOV/VLB1.0, H69/Adr, and HL60/AR). Furthermore, P-40 levels dropped significantly in one revertant cell line (H69/PR) derived from H69/AR cells. Interestingly, the expression of P-40 was also higher in two tumor cell lines (SKTax6a and A2780CP) that were selected with paclitaxel or cisplatin but do not express P-gp or MRP. Immuno-fluorescence staining of cells with IPM96 showed both membrane and cytoplasmic staining. These results were confirmed by Western blot analysis of different subcellular fractions from MCF-7/Adr cells. The membrane bound P-40 was resistant to extraction with high salt, chelating agents, and denaturing agents, but was solubilized with 10 mM CHAPS. Taken together, the overexpression of P-40 in multidrug resistant cells has not been previously determined and therefore could be important in the expression of the drug resistance phenotype.
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PMID:Overexpression of a 40-kDa protein in human multidrug resistant cells. 924 Apr 65

P-glycoprotein (P-gp), a plasma membrane glycoprotein associated with the multidrug resistance phenotype, is responsible for the ATP-dependent efflux of various amphiphilic drugs. Using membrane vesicles prepared from the multidrug resistant cell line DC-3F/ADX, we studied the perturbation of the basal (i.e. in the absence of drug) and verapamil-dependent P-gp ATPase activities induced by various detergents, at non-solubilizing, as well as at solubilizing, concentrations. The progressive membrane solubilization with increasing detergent concentration was monitored by light scattering and centrifugation experiments. For non-solubilizing detergent concentrations, all tested detergents except DOC induced a partial inhibition of P-gp ATPase activity, which was not correlated with the amount of the various tested detergents incorporated in the membranes. Analysis of the verapamil-induced P-gp activation reveals that P-gp ATPase activity is differently modulated by the various detergents at non-solubilizing concentrations. Thus, specific interactions between P-gp and detergents are more likely to occur rather than a global membrane perturbation. After solubilization by the various tested detergents, the basal P-gp ATPase activity was virtually completely inhibited, except in the presence of CHAPS which was able to preserve this activity at a level comparable to that measured in native membranes. However, the verapamil-induced P-gp ATPase activation was lost during P-gp solubilization by CHAPS, but recovered after dilution of CHAPS below its critical micellar concentration. These observations indicate specific interactions between P-gp and CHAPS molecules within the mixed micelles. On the whole, our data evidencing specific interactions P-gp/detergents are consistent with the location of the drug transport sites on P-gp transmembrane domains.
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PMID:Effects of detergents on P-glycoprotein atpase activity: differences in perturbations of basal and verapamil-dependent activities. 992 70