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)

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

Two new fused indoles were found to overcome multidrug resistance in P388/Adr cells in vitro. These agents potentiated the cytotoxicity of the antitumor drugs Adriamycin, vinblastine, and vincristine in multidrug-resistant cells with no effect on drug-sensitive parent P388 cells. They significantly increased the ATP-dependent accumulation of [3H]-vinblastine and inhibited efflux of the labeled drug from resistant cells. These compounds also inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine in P388/Adr cells and membranes isolated from these cells. In addition, the calcium antagonist activity of these compounds was very weak compared with that of verapamil. These data suggest that the compounds reported here may specifically overcome multidrug resistance without the serious hypotensive effects associated with calcium antagonists and that this activity may be independent of their ability to block calcium transport.
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PMID:Reversal of multidrug resistance by two novel indole derivatives. 135 8

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

A new combination of fluorescent dyes (rhodamine 123 and hydroethidine) was used to internally label hybridoma fusion partners. Murine hybridoma 520C9 (recognizing human c-erbB-2) was labeled with hydroethidine. Murine hybridoma 3G8 (recognizing human Fc gamma receptor III) was labeled with rhodamine 123, and verapamil was used to block rhodamine efflux via P-glycoprotein. Viability assays showed little cytotoxicity from these dyes at the concentrations used. The labeled cells were fused with polyethylene glycol, sorted for dual fluorescence on an Epics V cell sorter, and cloned. Hybrid hybridomas producing bispecific antibodies were selected for ability to promote lysis of SK-Br-3 breast cancer cells by human mononuclear cells. Several positive clones were obtained and shown to have a double content of DNA. Bispecific antibody produced by subclone 2B1 was purified by anion exchange chromatography and shown to bind both tumor cells and Fc gamma R III bearing cells. Using two parameter flow cytometric analysis, we were able to measure a 'bridging' effect of this bispecific antibody, which caused formation of complexes between PMNs and SK-Br-3 cells. Either parental antibody could compete with bispecific antibody to block such complexing. This fusion method provides several advantages over other techniques presently used (speed, convenience, low toxicity and automatic exclusion of dead cells) and can be applied to produce other hybrid hybridomas.
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PMID:Selection of hybrid hybridomas by flow cytometry using a new combination of fluorescent vital stains. 171 68

One of the characteristics of multidrug-resistant mammalian cells is the presence of a glycoprotein of approximately Mr 170,000 in their cell membrane. Termed P-glycoprotein, this glycoprotein is thought to be the gene product of an amplified gene cloned and sequenced from a cell line (LZ) which is highly resistant to Adriamycin and cross-resistant to actinomycin D, colchicine, and vincristine. Resistance to Adriamycin has been induced in sensitive cells by chromosome or gene transfer. We now show that P-glycoprotein isolated from LZ cells and fused to sensitive V79 Chinese hamster cells renders the latter transiently resistant to Adriamycin. Incorporation of P-glycoprotein was confirmed by immunoperoxidase staining of fusion products following treatment with antibody to P-glycoprotein and by Western blots of membrane preparations from fusion products. These results suggest that P-glycoprotein is one of the important factors in the expression of Adriamycin resistance and provide added confirmation that it may be the important product of gene amplification in multidrug-resistant cells. The results also suggest that the cell membrane may be one of several targets for Adriamycin cytotoxicity and that P-glycoprotein may be a binding site for Adriamycin, rendering the latter ineffective in registering sufficient membrane damage for cell killing.
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PMID:Transfer of adriamycin resistance by fusion of Mr 170,000 P-glycoprotein to the plasma membrane of sensitive cells. 196 29

We isolated revertant and resistant clones from multidrug-resistant K562/ADM cells and evaluated the expression of P-glycoprotein and the DNA copy number of MDR1. The 9 revertant clones contained 2- to 26-fold DNA copies of MDR1; however, they expressed an extensively decreased P-glycoprotein compared with K562/ADM, while the 10 multidrug-resistant clones contained 4- to 48-fold DNA copies, and the expression level of P-glycoprotein was dependent on the copy number of MDR1 DNA. The decreased expression of P-glycoprotein in the revertants was not due only to the loss of the copy number of MDR1 DNA. To elucidate the mechanism of P-glycoprotein expression decrease in the revertants, a revertant clone (R1-5) was fused with a multidrug-resistant clone (A2-1) or with a drug-sensitive clone isolated from K562. Compared with K562 clone, the A2-1 contained 32-fold MDR1 DNA copies and showed 131-fold resistance to Adriamycin. The revertant clone R1-5 contained 26-fold MDR1 DNA copies but expressed only 5% the P-glycoprotein of A2-1 cells and showed only 2-fold resistance to Adriamycin. For selection of intraspecific hybrids, a neomycin-resistant or a blasticidin S-resistant gene was introduced into clones by electroporation of pSV2neo or pSV2bsr. The introduction of these resistant genes did not alter the copy number or expression of MDR1 in the clones. Hybrid cells between R1-5bsr and A2-1neo were found to express 136 +/- 15% of the P-glycoprotein of A2-1 cells evaluated by quantitive flow cytometry. These hybrid cells contained 41- to 48-fold MDR1 copies and showed the multidrug-resistant phenotype, such as decrease of rhodamine123 accumulation and 120- to 210-fold resistance to Adriamycin (compared with K562), indicating that the 'silent' MDR1 genes in the revertant clone R1-5 were activated by cell fusion with an MDR clone. R1-5bsr x K562neo hybrids were found to contain 8- to 11-fold MDR1 copies and there was no increase in P-glycoprotein expression as compared with R1-5.
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PMID:Activation of silent MDR1 genes in revertant cells by fusion with multidrug-resistant cells. 749 79

The cDNA encoding the C-terminal nucleotide-binding domain (NBD2) from mouse P-glycoprotein involved in multidrug resistance was obtained from adrenal cell mRNA and amplified by reverse transcriptase polymerase chain reaction. NBD2 was highly overexpressed in Escherichia coli in fusion with glutathione S-transferase and could be purified after efficient thrombin cleavage. Both fused and purified NBD2 bound TNP (2',3'-O-(2,4,6-trinitrophenyl))- derivatives of nucleotides with high affinity. TNP-ATP or TNP-ADP binding at micromolar concentrations produced a characteristic blue-shifted enhancement of extrinsic fluorescence and was specifically prevented or chased by ATP or ADP at millimolar concentrations. A similar affinity binding was monitored by quenching of intrinsic fluorescence. The spectrum of fusion protein, containing 5 tryptophan residues, was maximally quenched at 328 nm upon interaction with TNP-nucleotides. TNP-GTP exhibited a lower affinity than TNP-ATP but produced a higher maximal quenching (44% instead of 28%). The intrinsic fluorescence of purified NBD2, containing a single tryptophan residue, exhibited a narrow spectrum with a maximum at 328 nm characteristic of a hydrophobic tryptophan environment. A high quenching was observed upon nucleotide interaction with similar affinity. The results put forward a functional role for the tryptophan-containing sequence of P-glycoprotein NBD2 that was not detected up to now.
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PMID:Overexpression and purification of the carboxyl-terminal nucleotide-binding domain from mouse P-glycoprotein. Strategic location of a tryptophan residue. 791 13

P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs. The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown. In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract. We have studied the tissue-specific expression of Caenorhabditis elegans P-glycoprotein genes pgp-1 and pgp-3 by transformation of nematodes with pgp-lacZ gene fusion constructs in which the promoter area of the pgp genes was fused to the coding region of lacZ. Expression of pgp-1 and pgp-3, as inferred from pgp-lacZ transgenic nematodes, was confined to the intestinal cells. The expression patterns of both genes were virtually indistinguishable. Quantitative analysis of pgp mRNA levels during development showed that pgp-1, -2, and -3 were expressed throughout the life cycle of C.elegans, albeit with some variation indicating developmental regulation. The expression of P-glycoprotein genes in intestinal cells is an evolutionarily conserved feature of these genes, consistent with the hypothesis that P-glycoproteins provide a mechanism of protection against environmental toxins.
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PMID:The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells. 809 15

The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol.
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PMID:ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB. 836 61


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