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 effectiveness of photodynamic therapy against P-glycoprotein ATPase activity in multidrug-resistant cells was studied. Chinese hamster ovary AUXB1 (drug-sensitive) and CR1R12 (multidrug-resistant) cell lines were compared with respect to uptake of 14C-polyhematoporphyrin and porphyrin photosensitization. Phototoxicity of Photofrin was similar in both cell lines, and no major differences in uptake or efflux of 14C-polyhematoporphyrin were observed. Porphyrin photosensitization in vitro of CR1R12 cells or isolated plasma membranes from these cells caused inhibition of P-glycoprotein ATPase activity. Application of porphyrin photosensitization at a sublethal level to CR1R12 cells resulted in a small but significant increase in adriamycin-induced cytotoxicity. The hydrophobic "picket-fence" porphyrin, meso-tetrakis-(o-propionamidophenyl)porphyrin, alpha,alpha,alpha,beta-isomer, was more inhibitory toward P-glycoprotein ATPase activity than the two less hydrophobic porphyrins tetraphenylporphine tetrasulfonate and Photofrin.
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PMID:Inhibition of the ATPase activity of P-glycoprotein by porphyrin photosensitization of multidrug-resistant cells in vitro. 774 83

P-glycoprotein is a membrane ATPase transporter responsible for multidrug resistance. Its primary structure is known from cDNA sequencing, but its tri-dimension structure remains hypothetical. Its physiological role in detoxification, as well as its involvement in anticancer drug transport, are no longer questioned, but its molecular mechanism of action remains unknown. It appears likely that it extrudes the drugs laterally, in the plane of the membrane, that several distinct drug or modulator binding sites exist, that its activity is regulated by phosphorylation. Purification and reconstitution of P-glycoprotein will probably allow to better understand its mechanism of action.
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PMID:[Structure and function of P-glycoprotein]. 774 17

The multidrug resistance gene product, P-glycoprotein or the multidrug transporter, confers multidrug resistance to cancer cells by maintaining intracellular levels of cytotoxic agents below a killing threshold. P-glycoprotein is located within the plasma membrane and is thought to act as an energy-dependent drug efflux pump. The multidrug transporter represents a member of the ATP-binding cassette superfamily of transporters (or traffic ATPases) and is composed of two highly homologous halves, each of which harbors a hydrophobic transmembrane domain and a hydrophilic ATP-binding fold. This review focuses on various biochemical and molecular genetic approaches used to analyze the structure, function, and mechanism of action of the multidrug transporter, whose most intriguing feature is its ability to interact with a large number of structurally and functionally different amphiphilic compounds. These studies have underscored the complexity of this membrane protein which has recently been suggested to assume alternative topological and quaternary structures, and to serve multiple functions both as a transporter and as a channel. With respect to the multidrug transporter activity of P-glycoprotein, progress has been made towards the elucidation of essential amino acid residues and/or polypeptide regions. Furthermore, the drug-stimulatable ATPase activity of P-glycoprotein has been established. The mechanism of drug transport by P-glycoprotein, however, is still unknown and its physiological role remains a matter of speculation.
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PMID:Molecular analysis of the multidrug transporter. 776 31

Multidrug-resistant tumor cells overexpress P-glycoprotein (170 kDa), a member of the ABC (ATP Binding Cassette)-transporter superfamily. P-glycoprotein has been implicated in transport of a broad range of amphiphilic, hydrophobic drugs from tumor cells. The sequence and structural organization of P-glycoprotein, which consists of 12 transmembrane helices and two cytoplasmic nucleotide binding domains, is similar to other ABC-transporters. It is believed that the nucleotide binding domains of various ABC transporters, which have 30-50% sequence identity, play an important role in coupling ATP hydrolysis to the transport process. To allow structure-function studies of the nucleotide binding domains, the carboxyl-terminal nucleotide binding domain (NBD) of Chinese hamster P-glycoprotein has been cloned, overexpressed, and purified both by itself and as a fusion with maltose-binding protein. It has been demonstrated that the carboxyl-terminal NBD, when overexpressed in Escherichia coli in the absence of transmembrane helices, has very low ATPase activity. This suggests that the amino-terminal nucleotide binding domain and possibly interaction with the transmembrane domains may be required for full ATPase activity. It is also consistent with the idea that the ATPase activity of P-glycoprotein is stimulated in the presence of drugs. Circular dichroism spectral analysis and the ability of carboxyl-terminal NBD, both by itself and as a fusion with maltose-binding protein, to bind ATP-agarose beads and P-glycoprotein specific monoclonal antibodies suggests that the polypeptide folds into a functional domain. Gel filtration chromatography and cross-linking studies indicate that the carboxyl-terminal NBD has a tendency to self-associate to form oligomers. It is speculated that the carboxyl-terminal NBD may play a role in self-association of P-glycoprotein molecules in the plasma membrane.
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PMID:Cloning, overexpression, purification, and characterization of the carboxyl-terminal nucleotide binding domain of P-glycoprotein. 777 70

The contribution of P-glycoprotein (Pgp) to multidrug resistance in human solid tumors is generally estimated from bulk mRNA measurements or immunohistochemistry, while direct measurement of the effect of Pgp on intracellular drug concentrations has not been pursued. We investigated the feasibility and sensitivity of a method for probing Pgp-mediated drug transport in cells isolated from solid tumors, using xenograft models. Human tumor xenografts (XG) were grown by s.c. injection of Pgp-expressing cell lines 2780AD, BRO/mdr1 and KB8-5. Tumor uptake of doxorubicin (DOX) after administration of DOX to the mice was determined. XG from untreated mice were enzymatically dissociated. The effect of the Pgp modulator bepridil on steady-state cellular daunorubicin (DNR) and vincristine (VCR) accumulation and chemosensitivity of these XG cells was compared with its effects in the cell lines (CL). mdr1 mRNA and Pgp (by flow cytometry) were measured. Also, the dependence on intracellular ATP concentration, [ATP]i, of the modulator effect was determined in intact KB8-5 cells. The results showed that i.v. administration of DOX to the mice led to lower DOX levels in the Pgp-expressing XG than in the "sensitive" XG, suggesting the presence of an in vivo functional Pgp in these XG tumor models. Dissociated, viable XG cells appeared to have ATP levels sufficient to sustain Pgp-ATPase-coupled drug transport. This was inferred from experiments using KB8-5 CL, which showed half-maximal inhibition of DNR transport at an [ATP]i of 1 to 2 mM. The effect of bepridil on DNR and VCR accumulation and chemosensitivity in the XG cells was in accordance with the XG expression of mdr1/Pgp. In KB8-5 XG cells, Pgp function was hardly detectable, in accordance with decreased mdr1/Pgp expression in vivo. In conclusion, Pgp activity can be determined in freshly dissociated XG human tumor cells. The results obtained with the more necrotic KB8-5 XG may represent some of the interpretation problems arising when low levels of Pgp expression occur within a heterogeneous cell population, such as may be expected in clinical human tumors. Also our results indicate that Pgp activity may be impaired in vivo at [ATP]i below 2 mM, which are realistic values for human solid tumors.
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PMID:Correlation between functional and molecular analysis of mdr1 P-glycoprotein in human solid-tumor xenografts. 779 Jan 25

Chinese hamster P-glycoprotein ("multidrug-resistance protein") was purified and reconstituted in proteoliposomes by the procedure of I. L. Urbatsch, M. K. al-Shawi, and A. E. Senior (1994, Biochemistry 33, 7069-7076). The presence of lipid during the octylglucoside solubilization and Reactive Red 120 chromatography steps was found to be mandatory for retention of ATPase activity. Sheep brain or bovine liver lipid extracts could be substituted for the Escherichia coli lipids used previously. Stimulation of ATPase activity of purified, reconstituted P-glycoprotein by vinblastine, colchicine, and daunomycin was seen with sheep brain and bovine liver lipids, but not with E. coli lipids. Basal (i.e., not drug-stimulated) ATPase activity was different in the three lipids. Azidopine labeling of the drug binding sites in purified, reconstituted P-glycoprotein was carried out; vinblastine, colchicine, and daunomycin competed for labeling in all three lipids. It is therefore evident that the lipid environment can significantly influence the characteristics of purified, reconstituted P-glycoprotein ATPase activity and the apparent coupling between drug-binding and catalytic sites.
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PMID:Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein. 784 Jun 7

The distribution of K(+)-ATPase activity in surface and crypt cells from rabbit distal colon was studied. Separation of surface and crypt cells was validated using the multidrug resistance gene (mdr 1) product, P-glycoprotein, as marker for differentiated surface epithelial cells. Western blot analysis revealed a 6-fold higher expression level of P-glycoprotein in colonic surface cells. K(+)-stimulated ouabain-insensitive ATPase activity was present in surface and in crypt cells. In surface cells, this K(+)-ATPase activity was only partly inhibitable by 10 microM SCH 28080, while in crypt cells K(+)-ATPase activity equalled SCH 28080-sensitive ATPase activity. These results strongly suggest the presence of two distinct K(+)-ATPases in colonic epithelial cells.
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PMID:Two distinct K(+)-ATPase activities in rabbit distal colon. 786 85

A single amino acid substitution, Gly185-->Val, in the human P-glycoprotein (Pgp) was previously shown to cause an altered pattern of drug resistance in cell lines transfected with the MDR1 cDNA carrying this mutation. To further define the function of amino acid 185 in the Pgp, the wild-type and the mutant Val185 Pgps were expressed in Sf9 insect cells, and their biochemical properties were compared. Verapamil- and colchicine-stimulated ATPase activities were markedly increased with concomitant increase in affinity for these compounds with Gly185-->Val substitution in the Pgp. However, the vinblastine-stimulated ATPase activities of the wild-type and Val185 Pgps were nearly identical. Because transport substrate-induced ATP hydrolysis is generally thought to reflect transport function, these data suggest that colchicine and verapamil are transported at an increased rate with Gly185-->Val substitution in the Pgp. These results also indicate that amino acid 185 is involved in verapamil and colchicine, but not in vinblastine, binding/transport. Kinetic analyses indicate that cyclosporin A, an inhibitor of Pgp, binds to the verapamil and vinblastine binding/transport site(s) in the Pgp. Taken together, the results presented herein reveal that the verapamil and vinblastine binding/transport site(s) are in close proximity and that the cyclosporin A binding site spans the common region of these two drug binding/transport site(s) in the Pgp molecule.
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PMID:Mutation of glycine 185 to valine alters the ATPase function of the human P-glycoprotein expressed in Sf9 cells. 789 10

P-glycoprotein, the multidrug transporter, is proposed to act as an ATP-driven drug efflux pump. We previously reported the partial purification of P-glycoprotein from multidrug-resistant cells (Doige, C. A., Yu, X., and Sharom, F. J. (1992) Biochim. Biophys. Acta 1109, 149-160). We now report the reconstitution of this preparation into phospholipid bilayers using rapid detergent removal by gel filtration chromatography. The resulting proteoliposomes displayed ATP-dependent [3H]colchicine uptake over a time period of 0-4 min. No drug uptake was observed for liposomes of lipid alone, or liposomes reconstituted with a similar extract from drug-sensitive cells. Drug uptake was osmotically sensitive, and abolished by detergent permeabilization, indicating that it represented true transport into the vesicle lumen. Steady-state levels of drug uptake increased with drug concentration, approaching saturation at approximately 150 microM colchicine, with half-maximal accumulation at 50 microM. Drug was accumulated actively against a 5.6-fold concentration gradient. Multidrug resistance spectrum drugs and chemosensitizers inhibited colchicine uptake by P-glycoprotein proteoliposomes, whereas cytosine arabinoside and methotrexate had no effect. Reconstituted liposomes showed high levels of ATPase activity, which was stimulated over 2-fold by verapamil and trifluoperazine. These results suggest that P-glycoprotein functions as an active drug transporter with constitutive ATPase activity.
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PMID:Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein. 790 Dec 14

Purified membrane vesicles were prepared from Chinese Hamster lung fibroblasts expressing high amounts of P-glycoprotein (P-gp), which is responsible for the multidrug resistance. P-gp ATPase activity, characterized in the presence or absence of verapamil, had a Michaelian behavior for its MgATP dependence. Thus only one MgATP molecule should be sufficient for the catalytic cycle. With increasing verapamil concentrations, a bell-shape curve was observed for ATPase activity, with half-activation and -inhibition concentrations of 1.2 microM and 490 microM, respectively. No cooperativity for verapamil was detected. These results strongly suggest that P-gp functions as an active transporter, with a coupling stoichiometry of one MgATP molecule hydrolysed for one verapamil molecule transported.
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PMID:Absence of cooperativity for MgATP and verapamil effects on the ATPase activity of P-glycoprotein containing membrane vesicles. 790 5


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