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)

With the goal of understanding possible mechanisms of drug resistance by the protozoan parasite Entamoeba histolytica (Eh), two novel Eh P-glycoprotein (Pgp) genes (Eh pgp5 and Eh pgp6) were sequenced, and the expression of four Eh pgp genes determined in wild-type (wt) clone A and emetine-resistant (EmR) clone C2 amebae. The Eh pgp5 gene encodes a 1301-amino acid (aa) protein that is similar to those of Eh pgp1 (64% aa identity), Eh pgp2 (61%), Eh pgp6 (39%) and Homo sapiens MDR (multidrug-resistance-encoding)(Hs MDR1; 38%) genes. The 1282-aa Eh pgp6 open reading frame (ORF), which is 19-28 aa shorter than those encoded by other Eh pgp, is also similar to those of Eh pgp1 (46% aa identity), Eh pgp2 (38%), and Hs MDR1 (39%). Both Eh pgp5 and Eh pgp6 ORF predict two ATP-binding cassettes and twelve hydrophobic alpha-helices, which form the putative transmembrane channel. EmR clone C2 amebae, growing at all concentrations of drug, show increased amounts of Eh pgp1 and Eh pgp6 mRNA when compared to wt clone A amebae. In contrast, only clone C2 amebae selected for growth at the highest concentrations of emetine (100-200 micrograms/ml) show increased Eh pgp5 mRNA, while mRNA of both clone C2 and clone A Eh amebae fail to bind an Eh pgp2-specific probe. It appears then that multiple Pgp may contribute to amebic Em resistance in vitro.
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PMID:Increase in mRNA of multiple Eh pgp genes encoding P-glycoprotein homologues in emetine-resistant Entamoeba histolytica parasites. 759 Mar 12

P-glycoprotein is an integral membrane protein that functions in multidrug resistance (MDR) cells as a drug efflux pump to maintain intracellular concentrations of antitumor drugs below cytotoxic levels. A homologue of the mammalian mdr gene has been isolated and characterized from Xenopus laevis (Xe-mdr). The cDNA was isolated from a tadpole cDNA library using the full length mouse mdrlb cDNA as a probe. The Xe-mdr encodes a protein that is 66% identical to the mouse mdrlb and 68% identical to the human mdrl. The predicted structure of the Xe-mdr gene product identifies twelve membrane spanning domains and two ATP binding sites both of which are the hallmark of the ABC (ATP binding cassette) transporters. Xe-mdr mRNA is expressed as a single message of 4.5 kb and is found predominantly in the intestine. Xe-mdr message is increased 3- to 4-fold in the ileum compared to the rest of the small intestine. In situ hybridization of sequential sections from the small intestine localized the expression of the Xe-mdr to the cells lining the lumenal epithelium. Brush border membrane vesicles prepared from the small intestine of Xenopus laevis effluxed vinblastine in an ATP-dependent manner. Efflux was decreased by verapamil, a known inhibitor of P-glycoprotein function. These studies indicate that the structure of Xe-mdr has been conserved and suggest that the protein has a role in maintaining the function of the normal intestine in Xenopus.
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PMID:A homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the intestine of Xenopus laevis. 759 85

P-glycoprotein confers multidrug resistance upon cells in which it is highly expressed, reducing the effectiveness of numerous cytotoxic drugs, including many of those used for chemotherapy of cancer. Although P-glycoprotein is widely believed to function as an ATP-dependent drug efflux pump, the unusually broad substrate specificity of P-glycoprotein has engendered the proposal of other, less direct mechanisms. None of the hypothetical mechanisms has been definitively tested, however, in a purified system where other cellular components and processes are absent. We have used a fluorescent substrate of P-glycoprotein, Hoechst 33342, to measure transport activity in real-time of highly purified P-glycoprotein in a reconstituted liposome system in which the P-glycoprotein has a uniformly inside-out orientation. Using this system, we demonstrated MgATP-dependent, chemosensitizer-inhibitable transport of Hoechst 33342. Transport was prevented by omission of Mg2+, by substitution of nonhydrolyzable adenylyl-beta,gamma-imidodiphosphate for ATP, by inhibition of the ATPase activity of P-glycoprotein with vanadate and N-ethylmaleimide, and by the chemosensitizers verapamil and amiodarone. Measurements of intraliposomal pH during Hoechst 33342 transport detected no large pH changes in P-glycoprotein-containing liposomes. These results are inconsistent with a mechanism in which P-glycoprotein affects drug accumulation by directly altering intracellular pH. The Hoechst 33342 transport assay results are consistent with mechanisms in which P-glycoprotein alone is sufficient to transport drugs out of the membrane bilayer.
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PMID:Reconstitution of drug transport by purified P-glycoprotein. 760 82

A known aporphine alkaloid, (-)-roemerine [1], isolated from the leaves of Annona senegalensis, was found to enhance the cytotoxic response mediated by vinblastine with multidrug-resistant KB-V1 cells. In the absence of vinblastine, no significant cytotoxicity was observed with KB-3 or KB-V1 cells (ED50 > 20 micrograms/ml), and several other human tumor cell lines were also relatively insensitive. As indicated by its ability to inhibit ATP-dependent [3H]vinblastine binding to multidrug-resistant KB-V1 cell membrane vesicles, (-)-roemerine appears to function by interacting with P-glycoprotein. In addition to alkaloid 1, three inactive compounds [the aporphine alkaloid(-)-isocorydine (reported in the levo-configuration for the first time), and the lignans (+/-)-8,8'-bisdihydrosiringenin [2] (a new natural product), and (+)-syringaresinol] were also isolated.
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PMID:(-)-Roemerine, an aporphine alkaloid from Annona senegalensis that reverses the multidrug-resistance phenotype with cultured cells. 762 38

In this study we report that the multidrug resistance protein (MRP) transports calcein from the cytoplasmic compartment of tumor cells, in contrast to P-glycoprotein which transports calcein acetoxymethyl ester from the plasmamembrane. The transport of calcein by MRP is ATP-dependent and is inhibited by probenecid and vincristine. Intracellular glutathione (GSH) depletion which occurred when cells were exposed to buthionine sulfoximine had no effect on the efflux of calcein, whereas it reversed the daunorubicin accumulation deficit in MRP overexpressing tumor cells. In conclusion, ATP-dependent transport of calcein and possibly other organic anions by MRP is not inhibited by a large decrease of the intracellular GSH concentration, that inhibits daunorubicin efflux by MRP.
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PMID:ATP-dependent efflux of calcein by the multidrug resistance protein (MRP): no inhibition by intracellular glutathione depletion. 762 44

P-Glycoprotein, the multidrug transporter, is isolated from the plasma membrane of CHRC5 cells using a selective two-step detergent extraction procedure. The partially purified protein displays a high level of ATPase activity, which has a high KM for ATP, is stimulated by drugs, and can be distinguished from that of other membrane ATPases by its unique inhibition profile. Delipidation completely inactivates ATPase activity, which is restored by the addition of fluid lipid mixtures. P-Glycoprotein was reconstituted into lipid bilayers with retention of both drug transport and ATPase activity. Proteoliposomes containing P-glycoprotein display osmotically sensitive ATP-dependent accumulation of 3H-colchicine in the vesicle lumen. Drug transport is active, generating a stable 5.6-fold concentration gradient, and can be blocked by compounds in the multidrug resistance spectrum. Reconstituted P-glycoprotein also exhibits a high level of ATPase activity which is further stimulated by various drugs. P-Glycoprotein therefore functions as an active drug transporter with constitutive ATPase activity.
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PMID:Characterization and functional reconstitution of the multidrug transporter. 762 46

The overexpression of the P-glycoprotein, the MDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [3H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.
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PMID:Purification and reconstitution of functional human P-glycoprotein. 762 47

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

The human multidrug resistance protein, or P-glycoprotein (Pgp), exhibits a high-capacity drug-dependent ATP hydrolytic activity that is a direct reflection of its drug transport capability. This activity is readily measured in membranes isolated from cultured insect cells infected with a baculovirus carrying the human mdr1 cDNA. The drug-stimulated ATPase activity is a useful alternative to conventional screening systems for identifying high-affinity drug substrates of the Pgp with potential clinical value as chemosensitizers for tumor cells that have become drug resistant. Using this assay system, a variety of drugs have been directly shown to interact with the Pgp. Many of the drugs stimulate the Pgp ATPase activity, but certain drugs bind tightly to the drug-binding site of the Pgp without eliciting ATP hydrolysis. Either class of drugs may be useful as chemosensitizing agents. The baculovirus/insect cell Pgp ATPase assay system may also facilitate future studies of the molecular structure and mechanism of the Pgp.
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PMID:Drug-stimulated ATPase activity of the human P-glycoprotein. 762 50

Chemotherapy, though it remains one of the front-line weapons used to treat human cancer, is often ineffective due to drug resistance mechanisms manifest in tumor cells. One common pattern of drug resistance, characterized by simultaneous resistance to multiple amphipathic, but otherwise structurally dissimilar anticancer drugs, is termed multidrug resistance. Multidrug resistance in various model systems, covering the phylogenetic range from bacteria to man, can be conferred by mammalian P-glycoproteins (PGPs), often termed multidrug transporters. PGPs are 170-kD polytopic membrane proteins, predicted to consist of two homologous halves, each with six membrane spanning regions and one ATP binding site. They are members of the ATP-binding cassette (ABC) superfamily of transporters, and are known to function biochemically as energy-dependent drug efflux pumps. However, much remains to be learned about PGP structure-function relationships, membrane topology, posttranslational regulation, and bioenergetics of drug transport. Much of the recent progress in the study of the human and mouse PGPs has come from heterologous expression systems which offer the benefits of ease of genetic selection and manipulation, and/or short generation times of the organism in which PGPs are expressed, and/or high-level expression of recombinant PGP. Here we review recent studies of PGP in E. coli, baculovirus, and yeast systems and evaluate their utility for the study of PGPs, as well as other higher eukaryotic membrane proteins.
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PMID:Heterologous expression systems for P-glycoprotein: E. coli, yeast, and baculovirus. 762 51


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