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

Two ATP-binding domains are found in members of the family of ATP-dependent transport proteins, which includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, full-length and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the Escherichia coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprotein beta-galactosidase showed ATPase activity with apparent specific activity of 180 nmol/mg/min, a value higher than previously reported, in the presence of phospholipids, suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain.
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PMID:P-glycoprotein. ATP hydrolysis by the N-terminal nucleotide-binding domain. 134 41

We fused P-glycoprotein with beta-galactosidase at the C-terminus aiming to study the mechanism of drug binding of P-glycoprotein in reconstitution experiments. Expression of the fusion protein in NIH 3T3 cells conferred a multidrug-resistant phenotype, suggesting that beta-galactosidase fusion at the C-terminus does not affect the functions of P-glycoprotein. The fusion protein was partially purified by simple immunoprecipitation with anti-beta-galactosidase polyclonal antibody, and its [3H]azidopine binding property was investigated in the presence of various compositions of liposomes. The purified P-glycoprotein, after reconstitution into liposomes, was capable of binding [3H]azidopine. When the cholesterol content of liposomes was increased to a weight ratio of 20%, the specific binding activity of the partially purified fusion protein was stimulated, and when the cholesterol content was increased higher, the binding activity decreased. The binding was specifically decreased by competition with vinblastine. Stigmasterol was less effective, and ergosterol was the least effective in stimulating the specific binding.
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PMID:Specific drug binding by purified lipid-reconstituted P-glycoprotein: dependence on the lipid composition. 135 29

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.
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PMID:Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein. 136 54

The multiple drug-resistant human lymphoblastic leukemic cell, CEM/VLB100, in which P-glycoprotein (P-170) is overexpressed, has a lowered content of lysosomal enzymes, such as N-acetylglucosaminidase and beta-galactosidase, and the relative rates of secretion of these enzymes are significantly greater than those of its drug-sensitive counterpart, CEM. The ability of CEM/VLB100 cells to accumulate [3H]vinblastine ([3H]-VLB) is also greatly reduced. Multiple drug-resistant cells whose mode of resistance is not associated with P-170 do not have reduced enzyme content, and their rate of secretion is the same as that of their drug-sensitive parents. Linkage of drug and enzyme elimination is suggested by the observation that verapamil inhibits both the efflux of [3H]VLB and the secretion of lysosomal enzymes in CEM/VLB100 cells; the content of both [3H]VLB and enzyme increases in these cells when chronically exposed to verapamil. Further, both secretion of N-acetylglucosaminidase and efflux of [3H]VLB by CEM/VLB100 cells are enhanced by the addition of NaCl to the suspending, sucrose-containing medium. When cells have taken up [3H]VLB and are then fractionated by means of a Percoll centrifugation gradient, the distribution of drug among the various populations of vesicles is similar to that of N-acetylglucosaminidase. Losses of both enzyme and drug take place from these vesicular populations to varying degrees, when CEM/VLB100 cells are induced to secrete. It is proposed that, in a multiple drug-resistant cell such as CEM/VLB100, the presence of P-170 in the plasma membrane may, in some indirect manner, lead to increased exocytosis of lysosomal enzyme, ultimately resulting in a significant depletion of enzyme. Further, a toxic, cationic drug such as vinblastine, accumulating in lysosomes and acidic vesicles, is also eliminated from the cell by exocytosis. This pathway may supplement the known, major mode of efflux directly involving P-170.
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PMID:Secretion of lysosomal enzymes by drug-sensitive and multiple drug-resistant cells. 167 22

The MDR1 gene product P-glycoprotein (P-gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rh123). Modulation of the level of P-gp expression has the potential of overcoming multidrug resistance. One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells. The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rh123 extrusion was used to test P-gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit. Human CD34+ selected cells were analysed immediately following transduction. In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P-gp expressing Rh123 dull cells. To monitor transduction efficiency over time as well as the possibility of in vivo selection of drug-resistant BM cells in mice treated with increasing numbers of taxol cycles, the assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rh123 efflux in transduced cells. Analysis of another fluorescence assay using fluorescein di-beta galactopyranoside as a substrate for beta-galactosidase in cells transduced with a MDR1: beta-gal activity. We conclude that the Rh123 efflux assay is a sensitive method to monitor P-gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rh123 dull cells.
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PMID:Transduction of MDR1 into human and mouse haemopoietic progenitor cells: use of rhodamine (Rh123) to determine transduction frequency and in vivo selection. 766 66

Multidrug resistance (MDR) can be conferred by overexpression of the adenosine triphosphate-driven multidrug transporter P-glycoprotein (Pgp) known as MDR1. Thus, two potential applications of the MDR1 gene that may be useful in gene therapy are the protection of bone marrow cells from the cytotoxic effects of chemotherapy regimens in cancer patients and its possible use as an in vivo selectable gene when linked to a therapeutic gene. In this study, we have designed two retroviral bicistronic expression vectors by linking the MDR1 gene to the reporters known as beta-galactosidase and the red-shifted green fluorescent protein (GFP). We report the creation of stable producer cell lines that synthesize virus particles carrying the MDR-internal ribosomal entry site (IRES)-lacZ and the MDR-IRES-GFP transgenes. These transcriptional fusions allow coordinate expression of Pgp and the reporter gene product to easily mark the MDR phenotype. Using the MDR-IRES-lacZ retrovirus, we demonstrate that periodic pulses of cytotoxic drug selection with a Pgp substrate enable sustained, long-term expression of the reporter beta-galactosidase in otherwise unstable transductants. We have also incorporated the improved features of GFP as a reporter gene into our MDR-IRES-GFP retrovirus. This vector allows rapid and specific identification of MDR1 gene transfer and expression in living cells either by fluorescence microscopy or by fluorescence-activated cell sorter analysis. These two MDR/reporter gene systems should be useful for in vivo studies, the evaluation of the potential of the MDR1 gene in gene therapy applications, and as a monitor of the selective efficacy of its MDR phenotype.
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PMID:Construction and characterization of bicistronic retroviral vectors encoding the multidrug transporter and beta-galactosidase or green fluorescent protein. 969 71