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)

Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).
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PMID:Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. 135 64

To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells.
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PMID:Volume-sensitive chloride channel activity does not depend on endogenous P-glycoprotein. 749 63

A 160-kDa plasma membrane protein of the yeast Saccharomyces cerevisiae was overexpressed by mutating the PDR1 or the PDR3 transcription factor gene. The protein is the membrane-bound ATP binding cassette transporter PDR5 (Balzi, E., Wang, M., Leterme, S., Van Dyck, L., and Goffeau, A. (1994) J. Biol. Chem. 269, 2206-2214). PDR5 was solubilized with n-dodecyl-beta-D-malto-side and separated from the PMA1 plasma membrane H(+)-ATPase by glycerol gradient centrifugation. The PDR5 protein hydrolyzes nucleoside diphosphates and triphosphates. This activity is sensitive to low concentrations of vanadate, of oligomycin, and of a variety of hydrophobic compounds. Many of these properties liken PDR5 to the purified mammalian P-glycoprotein responsible for multidrug resistance.
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PMID:Solubilization and characterization of the overexpressed PDR5 multidrug resistance nucleotide triphosphatase of yeast. 817 92

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

To clarify renal handling of rhodamine 123, a substrate for P-glycoprotein, in normal and diseased states, in-vivo clearance studies were performed with normal rats and rats with glycerol-induced acute renal failure. For normal rats the excretion ratio of unbound rhodamine 123-to-inulin was 3.25, indicating the presence of the renal tubular secretion of rhodamine 123. Co-administration of cyclosporin, a P-glycoprotein inhibitor, significantly reduced tubular secretion of rhodamine 123. Administration of glycerol induced both an increase in blood urea nitrogen and a reduction in the glomerular filtration rate, confirming the induction of acute renal failure. Total plasma, renal, and tubular secretory clearances of rhodamine 123 were significantly lower for rats with acute renal failure than for control rats. There was no difference between the ATP content of the renal cortex in control rats and those with acute renal failure. In addition to the decrease in renal clearance, a decrease in the biliary clearance of rhodamine 123 was also observed in rats with acute renal failure. These results imply that rhodamine 123 is secreted via P-glycoprotein in renal tubules and that the renal secretory clearance of rhodamine 123 was reduced after acute renal failure, probably because of impairment of P-glycoprotein.
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PMID:Renal excretion of rhodamine 123, a P-glycoprotein substrate, in rats with glycerol-induced acute renal failure. 982 64

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies.
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PMID:Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. 1072 88

The effect of glycerol-induced acute renal failure on P-glycoprotein expression and function was evaluated in rats. The in vivo function of P-glycoprotein was evaluated by measuring renal secretory and biliary clearance and brain distribution of rhodamine 123 (Rho-123), a P-glycoprotein substrate, under a steady-state plasma concentration. In acute renal failure rats, the P-glycoprotein level increased 2.5-fold in the kidney, but not in the liver and brain. In contrast, P-glycoprotein function in these tissues was suppressed. Interestingly, not only the renal but also the biliary clearance of Rho-123 was correlated with the glomerular filtration rate. In Caco-2 cells, plasma from renal failure rats exhibited a greater inhibitory effect on P-glycoprotein-mediated transport of Rho-123 than did plasma from control rats. In conclusion, P-glycoprotein function was systemically suppressed in acute renal failure, even though the level of P-glycoprotein remained unchanged or rather increased. This may be due to the accumulation of some endogenous P-glycoprotein substrates/modulators in the plasma in disease states.
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PMID:Expression and function of P-glycoprotein in rats with glycerol-induced acute renal failure. 1104 Mar 53

The purpose of this work was to investigate if P-glycoprotein (Pgp) efflux pump activity could be inhibited in the sub-bronchial epithelial cell line, Calu-3, by glucocorticosteroids and beta-ligands. The Pgp modulation efficiency of each compound was determined by its ability to increase the accumulation of the Pgp substrate rhodamine 123 (Rh123) accumulation in these cells. Pgp inhibition was observed at > or =100 microM steroids and beta-ligand. The modulation effectiveness of the beta-ligands increased with increasing hydrophobicity (logP(octanol/aqueous)) whereas an obvious correlation was not obtained with the complete set of steroids tested. Steroidal Pgp substrates did not affect Rh123 accumulation (e.g. aldosterone, dexamethasone, 11beta,17alpha,21-OH progesterone). In contrast, two hydrophobic non-Pgp steroidal substrates (testosterone and progesterone) displayed different effects on Rh123 accumulation, with progesterone being the more potent modulator. The most hydrophobic beta-ligand, propranolol, a known Pgp substrate, gave the largest increase in Rh123 accumulation in this therapeutic class. The beta-ligand modulation efficiency could also be correlated to Pgp structural recognition elements such as hydrogen bonding potential, the presence of a basic nitrogen and planar aromatic ring. No effect on Rh123 accumulation was observed with the formulation additives tested (ethanol, glycerol and palmitoyl carnitine) at concentrations previously reported to be non-toxic to Calu-3 cells.
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PMID:Modulation of P-glycoprotein activity in Calu-3 cells using steroids and beta-ligands. 1157 79

The expression and function of P-glycoprotein (P-gp), an ATP-dependent efflux pump, were examined in rats pretreated with dexamethasone (DEX), an inducer of P-gp, and in rats with glycerol-induced acute renal failure (ARF) and with CCl4-induced acute hepatic failure (AHF). DEX pretreatment increased the P-gp level and its functional activity in the intestine. In contrast, in ARF and AHF rats, the in vivo P-gp function was systemically suppressed, even though the level of P-gp remained unchanged or rather increased. In Caco-2 cells, the plasma collected from diseased rats exhibited a greater inhibitory effect on P-gp function than did plasma from control rats. A higher-plasma level of corticosterone, an endogenous P-gp substrate/inhibitor, was observed in the disease rats. These findings indicate that the actual in vivo function of P-gp cannot be predicted merely from the expression level of P-gp, and suggest that some endogenous P-gp-related compounds such as corticosterone participate in the regulation of in vivo P-gp function in diseased states.
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PMID:Factors affecting the expression and function of P-glycoprotein in rats: drug treatments and diseased states. 1187 84

Studies of the multidrug-resistance protein 1 (MRP1) have been hampered by the lack of a simple expression system allowing for rapid generation of mutants and yielding milligram amounts of protein. Here, we describe a Saccharomyces cerevisiae expression system that meets those conditions. MRP1 was expressed under the control of the constitutive PMA1 (yeast proton pump) promoter. The best conditions for expression were determined, including the use of the chemical chaperone glycerol, which increased MRP1 expression. N-terminal poly-histidine or FLAG affinity tags reduce MRP1 expression, whereas the same tags fused to the C-terminus had no effect. All the fusion proteins were functional. We conclude that because of its low cost and simplicity, the S. cerevisiae-based MRP1-expression system will be useful for studies where a large number of mutants or milligram amounts of purified MRP1 are needed.
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PMID:Expression of functional multidrug-resistance protein 1 in Saccharomyces cerevisiae: effects of N- and C-terminal affinity tags. 1281 67


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