Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an N(in)-C(out) rather than the predicted N(out)-C(in) orientation, and TM3 is in cytoplasm rather than the predicted N(in)-C(out) orientation in the membrane. It is possible that TM4 has a strong activity to initiate the N(in)-C(out) membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo N(in)-C(out) membrane insertion activity whereas TM4 initiates the N(in)-C(out) membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates N(in)-C(out) membrane insertion whereas TM4 stops the insertion event; and 2) "leaving one TM segment out of the membrane" may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the N(in)-C(out) membrane insertion activities of the TM segments.
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PMID:Dissection of de novo membrane insertion activities of internal transmembrane segments of ATP-binding-cassette transporters: toward understanding topological rules for membrane assembly of polytopic membrane proteins. 952 83

Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump. Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents. The mechanism of coupling of ATP hydrolysis to drug transport is not known. To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane. In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography. Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (specific activity ranging from 4.5 to 6.5 micromol min-1 mg-1). The ATPase activity was inhibited by ADP in a competitive manner, and by vanadate and N-ethylmaleimide at low concentrations. Vanadate which is known to inhibit ATPase activity by trapping MgADP at the catalytic site inhibited photoaffinity labeling of Pgp with substrate analogues, [125I]iodoarylazidoprazosin and [3H]azidopine, only under ATP hydrolysis conditions. Because vanadate-trapped Pgp is known to resemble the ADP and phosphate-bound catalytic transition state, our findings indicate that ATP hydrolysis results in a conformation with reduced affinity for substrates. A catalytic transition conformation with reduced affinity would essentially result in substrate dissociation and supports a model for drug transport in which an ATP hydrolysis-induced conformational change leads to drug release toward the extracellular medium.
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PMID:Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state. 953 20

The development of multidrug resistance (MDR) in heterogeneous cell sensitive and resistant populations to a variety of clinically important cytotoxic drugs poses a major obstacle to cancer chemotherapy. The MDR phenotype is characterized by a decrease the intracellular drug accumulation and by an overexpression of the MDR1 gene which encodes the membrane protein, P-glycoprotein (Pgp). To evaluate the MDR phenotype, rationale investigations of the cytotoxic processes and effect,s of Adriamycin (ADR) were done to obtain information on individual cells. Such information could be obtained through a multiparametric approach involving multiwavelength microfluorometry and numerical image analysis on single living cells. To achieve this, cells should be simultaneously stained with Hoechst 33342 (nuclear staining), Rhodamine 123 (mitochondria staining) and Nile Red (cell contour delineation). Changes in the biological parameters accessible from R123, Ho33342 and C-SNARF-1/AM (probe used for the pHi measurements) labelling were found more informative than changes in morphological parameters for the discrimination of sensitive and resistant cells. Furthermore, this approach allows the discrimination between two resistant cell lines expressing different mechanisms of resistance.
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PMID:Multiwavelength videomicrofluorometric study of some human leukemic lymphoblasts: effect of adriamycin on some biological parameter. 961 71

P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp.
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PMID:Secondary structure of P-glycoprotein investigated by circular dichroism and amino acid sequence analysis. 963 Jul 1

P-Glycoprotein, the plasma membrane protein responsible for the multidrug resistance of some tumour cells, is an active transporter of a number of structurally unrelated hydrophobic drugs. We have characterized the modulation of its ATPase activity by a multidrug-resistance-related cytotoxic drug, vinblastine, and different multidrug-resistance-reversing agents, verapamil and the dihydropyridines nicardipine, nimodipine, nitrendipine, nifedipine and azidopine. P-Glycoprotein ATPase activity was measured by using native membrane vesicles containing large amounts of P-glycoprotein, prepared from the highly multidrug-resistant lung fibroblasts DC-3F/ADX. P-Glycoprotein ATPase is activated by verapamil and by nicardipine but not by vinblastine. Among the five dihydropyridines tested, the higher the hydrophobicity, the higher was the activation factor with respect to the basal activity and the lower was the half-maximal activating concentration. The vinblastine-specific binding on P-glycoprotein is reported by the inhibitions of the verapamil- and the nicardipine-stimulated ATPase. These inhibitions are purely competitive, which means that the bindings of vinblastine and verapamil, or vinblastine and nicardipine, on P-glycoprotein are mutually exclusive. In contrast, verapamil and nicardipine display mutually non-competitive interactions. This demonstrates the existence of two distinct specific sites for these two P-glycoprotein modulators on which they can bind simultaneously and separately to the vinblastine site. The nicardipine-stimulated ATPase activity in the presence of the other dihydropyridines shows mixed-type inhibitions. These dihydropyridines have thus different binding sites that interact mutually to decrease their respective, separately determined affinities. This could be due to steric constraints between sites close to each other. This is supported by the observation that vinblastine binding is not mutually exclusive with nifedipine or nitrendipine binding, whereas it is mutually exclusive with nicardipine. Moreover, verapamil binding also interacts with the five dihydropyridines by mixed inhibitions, with different destabilization factors. On the whole our enzymic data show that P-glycoprotein has distinct but interacting binding sites for various modulators of its ATPase function.
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PMID:Multidrug resistance transporter P-glycoprotein has distinct but interacting binding sites for cytotoxic drugs and reversing agents. 965 75

The sister gene of P-glycoprotein (Spgp) is a liver-specific ATP-binding cassette protein highly related to the P-glycoprotein (Pgp) family (S. Childs et al, Cancer Res., 55: 2029-2034, 1995). Spgp appears to be related to the Pgp family by an ancient duplication occurring before the division of fish and mammals. P-Glycoproteins have diverse functions including broad specificity multidrug resistance in cell lines and tumors, detoxification of tissues such as the intestine and blood-brain barrier, and phosphatidylcholine transport in liver. Spgp is a Mr approximately 170,000 glycosylated plasma membrane protein localized to the canalicular surface of hepatocytes in the rat liver. The full-length cDNA of Spgp was isolated from rat, and its expression was characterized in situ and in transfected cells. The expression of Spgp correlates with the differentiation of hepatocytes and is seen only in late liver development. It is not observed in hepatoma cell lines. The physiological function of Spgp in liver is unknown, but it maps to 2q31 in humans, in the vicinity of liver transport disorders for bile acids and cholesterol. Spgp may therefore be involved in some aspect of bile acid or cholesterol metabolism. Spgp transfectants have a low level resistance to Taxol but not to other drugs that form part of the multidrug resistance phenotype. This resistance is reversible by the Pgp-reversing agents cyclosporin A, PSC833, and verapamil, suggesting a conservation in some functions of Pgps across large evolutionary distance.
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PMID:Taxol resistance mediated by transfection of the liver-specific sister gene of P-glycoprotein. 975 29

We generated Chinese hamster ovary cells which are highly multidrug-resistant by selection in colchicine. Purified plasma membranes from these cells are enriched in P-glycoprotein (Pgp), up to 32% w/w of membrane protein. From plasma membranes we purified Pgp to homogeneity and reconstituted it in proteoliposomes. Both plasma membranes and purified reconstituted Pgp show drug-stimulated ATPase activity (approximately 20 s-1), comparable to other transport ATPases. These materials enable investigation and characterization of the catalytic sites and mechanism. Various approaches have been used, notably enzyme kinetics, photoaffinity and other covalent labelling, use of vanadate as transition-state analog, and inhibition by beryllium and aluminum fluoride. Both Pgp nucleotide sites hydrolyse MgATP and are of relatively low specificity and affinity for nucleotides. Trapping of nucleotide by vanadate in either site blocks catalysis at both sites; covalent inactivation of either site completely blocks turnover. Therefore the catalytic sites interact strongly, and it appears that when one site enters the transition-state conformation the other site is prohibited from doing so. A minimal reaction scheme for ATP hydrolysis has been determined. We have proposed an alternating catalytic sites scheme, in which drug-transport is coupled to relaxation of a high chemical potential conformation of the catalytic site (Pgp.MgADP.Pi) which is generated by the hydrolysis step itself. Photoaffinity labelling of Pgp catalytic sites has revealed equivalent Tyr residues which lie close to the adenine ring of bound MgATP in both sites.
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PMID:Catalytic mechanism of P-glycoprotein. 978 63

P-glycoprotein (P-gp), encoded by the mdr1a gene, is an ATP-dependent plasma membrane protein that is expressed in abundance on the blood-brain barrier (BBB). P-gp limits the CNS influx and retention of a variety of lipophilic compounds. We hypothesized that brain bilirubin content after an i.v. bilirubin infusion would be increased in P-gp-deficient mdr1a null mutant transgenic mice (mdr1a(-/-)) compared with controls. Eighteen mdr1a(-/-) null mutant and 18 P-gp-sufficient wild type mice (+/+) were anesthetized and 50 mg/kg bilirubin infused through the tail vein. Brain bilirubin content (mean +/- SEM) 10 min after infusion was significantly higher in mdr1a(-/-) (18.1 +/- 2.4 nmol/g) compared with (+/+) mice (10.4 +/- 1.0 nmol/g). Brain bilirubin content declined 60 min after infusion but remained higher in mdr1a(-/-) (10.3 +/- 1.4 nmol/g) compared with (+/+) mice (5.3 +/- 0.9 nmol/g). Brain bilirubin clearance did not differ between groups (t 1/2 approximately 55 min). We conclude that P-gp-deficient mdr1a(-/-) mice have significantly higher brain bilirubin content compared with controls after an i.v. bilirubin load. These data suggest that 1) bilirubin is a substrate for P-gp and 2) the increased brain bilirubin content in mdr1a(-/-) mice is due to enhanced brain bilirubin influx. We speculate that BBB P-gp provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.
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PMID:Brain bilirubin content is increased in P-glycoprotein-deficient transgenic null mutant mice. 980 59

One important mechanism of drug resistance in acute leukemia is the overexpression of the multi-drug resistance (MDR1) gene that encodes a 170-kDa membrane protein called P-glycoprotein. To estimate the incidence and role of MDR1 gene expression in patients with acute leukemia, we investigated the expression of MDR1 by using the RT-PCR method in blast cells from 40 cases of de novo acute leukemia. We found a high frequency of MDR1 gene expression: 10 out of 20 with de novo acute myeloid leukemia (AML), 8 out of 17 with de novo acute lymphoblastic leukemia (ALL), and none of the 3 with de novo acute mixed leukemia, were MDR1 mRNA-positive. No correlation between cluster designation (CD) surface markers (CD19, CD7, CD13, CD33, CD34, CD14, HLA-DR) and MDR1 gene expression in AML was found. The complete remission rate was correlated with MDR1 gene expression. Among 40 evaluable patients examined, 17% (3 of 18) with MDR1 mRNA-positive reached complete remission versus 77% (17 of 22) with MDR1 mRNA-negative (p=0.044). These results suggest that MDR1 gene expression can be used as a prognostic factor and may be helpful in determining chemotherapeutic protocol for patients with acute leukemia.
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PMID:Multi-drug resistance (MDR1) gene expression in de novo acute leukemia cells: correlations with CD surface markers and treatment outcome. 988 70

P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport [Shapiro, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 130-137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by [125I]iodoarylazidoprazosin [Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry 33, 256-265] and is likely not to be capable of drug transport.
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PMID:Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone. Evidence for a third drug-binding site. 1009 72


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