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)

In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.
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PMID:ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. 135 66

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.
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PMID:Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein. 196 66

The Mr 170,000 to 180,000 membrane glycoprotein associated with multidrug resistance (P-glycoprotein) is involved in drug transport mechanisms across the plasma membrane of multidrug-resistant cells. We have recently reported the purification of P-glycoprotein. The purified P-glycoprotein was found to have an ATPase activity, which might be coupled with the active efflux of anticancer drugs. In the present study, we have further studied the properties of the P-glycoprotein ATPase activity by an immobilized enzyme assay procedure using a P-glycoprotein-antibody-Protein A-Sepharose complex. GTP was also hydrolyzed by the P-glycoprotein, although less efficiently than ATP. The ATPase activity of P-glycoprotein had an optimal pH range around neutrality (pH 6.5-7.4). The detergent concentration of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate used for protein solubilization was essential for enzyme recovery. Maximum activity was obtained when 0.1-0.2% 3-[(3-cholamidopropyl)dimethyl-ammonio]-propane sulfonate was used, while higher concentrations markedly inhibited the ATPase activity. The ATPase activity was dependent on Mg2+; maximum activity was obtained at 2-10 mM. Manganese and cobalt could substitute for magnesium as ionic cofactors. Divalent cations such as Ca2+, Zn2+, Ni2+, Cd2+, and Cu2+ inhibited the Mg2+-catalyzed ATP hydrolysis. N-Ethylmaleimide and vanadate inhibited the ATPase activity, while sodium azide or ouabain had no effect. Anticancer agents such as vincristine and Adriamycin did not affect the enzyme activity. In contrast, verapamil and trifluoperazine, agents which inhibit active drug efflux and restore drug sensitivity in resistant cells, caused an increase in the P-glycoprotein ATPase activity suggesting that P-glycoprotein might be the target molecule of these agents.
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PMID:Characterization of the ATPase activity of the Mr 170,000 to 180,000 membrane glycoprotein (P-glycoprotein) associated with multidrug resistance in K562/ADM cells. 290 Jun 77

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

We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 [Lelong et al. (1992) FEBS Lett. 304, 256-260]. Like [gamma-32P]ATP, [gamma-32P]GTP was also able to phosphorylate Pgp in vitro. Unlabeled GTP enhanced the phosphorylation of the transporter by [gamma-32P]ATP, whereas unlabeled ATP inhibited incorporation of label. While phosphorylation by [gamma-32P]ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+. Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation. The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter. Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides. These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.
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PMID:GTP-stimulated phosphorylation of P-glycoprotein in transporting vesicles from KB-V1 multidrug resistant cells. 791 30

We designed a hammerhead ribozyme which site-specifically cleaved the GUC sequence in codon 179 of MDR1 mRNA. The cleavage site was 6 amino acids upstream from the drug binding site and was considered sufficiently close to the essential locus for P-glycoprotein function. The ribozyme cleaved the MDR1 mRNA under physiological conditions in vitro. The cleavage was dependent on ribozyme concentration and on incubation time. Mg2+ ion was essential for the cleavage. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.
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PMID:Cleavage of human MDR1 mRNA by a hammerhead ribozyme. 809 9

Generation of bile flow is a regulated, ATP-dependent process and depends on the coordinated action of a number of transporter proteins in the sinusoidal and canalicular domains of the hepatocyte. Dysfunction of any of these proteins leads to retention of substrates, with conjugated hyperbilirubinemia or cholestasis as a result. In recent years many of the transport proteins involved in bile formation have been identified, cloned, and functionally characterized. The hepatocyte sinusoidal membrane contains transport proteins for the hepatic uptake of organic anions and cations and for the uptake of bile acids. The multispecific organic anion transporting polypeptide (OATP) mediates the hepatic uptake of organic anions and a variety of organic amphiphilic compounds, including organic cations. The organic cation transporter OCT1 more specifically transports small organic cations. NTCP is the Na(+)-bile acid cotransporting protein that mediates the hepatic uptake of bile acids. The canalicular transport proteins are able to transport endogenous and exogenous metabolites into the bile against steep concentration gradients. Most of these transporters are members of the large ATP-binding cassette (ABC) superfamily, and their transport function directly depends on the hydrolysis of Mg2+/ATP. At least five ABC transporter proteins have been characterized so far: 1) the human multidrug resistance protein MDR1 mediates the excretion of hydrophobic, mostly cationic, metabolites; 2) MDR3 is involved in phosphatidylcholine secretion; 3) the canalicular bile acid transporter cBAT mediates secretion of monovalent bile salts and provides the molecular basis of bile acid-dependent bile flow; 4) SPGP, product of the P-glycoprotein sister gene, is exclusively expressed in the liver but its function is currently unknown; and 5) the human multidrug resistance protein MRP2 mediates the excretion of multivalent anionic conjugates.
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PMID:Molecular aspects of hepatobiliary transport. 922 63

P-glycoprotein (Pgp) mediates drug transport utilizing the energy released from ATP hydrolysis. However, the mechanism by which Pgp couples these two reactions remains unclear. The present work is undertaken to describe kinetically the first step, which is the interdependence of nucleotide and drug binding to the Pgp by the use of vanadate. Preincubation of human Pgp expressed in Sf9 insect cells with vanadate in the presence of Mg2+, ATP, and verapamil resulted in nearly complete and stable inhibition of the drug-stimulated ATPase function. In contrast, the Pgp ATPase function was nearly unaffected when Mg2+, ATP, or verapamil was omitted. Inhibition was highly specific for divalent cations that support ATP hydrolysis, for nucleotides that serve as substrates of hydrolysis, and for those drugs/compounds that interact with the drug-binding/transport sites of the Pgp. Kinetic analysis indicated that vanadate inhibition was MgATP concentration-dependent with an apparent Ki value similar to the apparent Km, suggesting that MgATP was bound to a similar ATP-binding site in both the ATPase inhibition and activation reactions. In support of this conclusion, vanadate, in the presence of Mg2+ and verapamil, caused selective trapping of 8-azido [alpha-32P] ATP and covalent labeling of ATP-binding site in the Pgp. Differences were observed in the vanadate-induced inhibition of wild-type and Val185 mutant Pgp's with different drug/compounds. These results suggested that the affinity of the interacting drug/compound is a constant and influences the overall stability of the inhibited Pgp species. Possible implications of these observations for the coupling of ATP hydrolysis to drug transport are discussed.
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PMID:Drug binding and nucleotide hydrolyzability are essential requirements in the vanadate-induced inhibition of the human P-glycoprotein ATPase. 977 76

Doxorubicin, a drug largely used in chemotherapy, is transported by P-glycoprotein, a protein involved in the multidrug-resistance phenotype. Taking advantage of the doxorubicin fluorescence quenching upon interaction with DNA, a sensitive assay of this active transport can be carried out: quantitative in vitro studies could be achieved with DNA-loaded proteoliposomes, after correction for the doxorubicin passive diffusion through phospholipids. In this paper, we describe experimental conditions that will be relevant to P-glycoprotein studies. Efficient DNA entrapment in preformed liposomes was obtained using the freeze/thawing procedure, and the doxorubicin passive diffusion was quantified in the presence of ATP/Mg2+, the second substrate of P-glycoprotein. The doxorubicin diffusion rate decreases in the presence of ATP, indicating an interaction between doxorubicin and ATP that will hinder any measurement of ATP-driven transport. The interaction between doxorubicin and ATP was studied by fluorescence quenching, octanol/buffer partition coefficient, and diffusion rate into DNA-loaded liposomes. The results give evidence for complex interactions. However, under our experimental conditions, these interactions are only slightly modified in the presence of Mg2+. Since this cation is essential for P-glycoprotein activity, it can be concluded that in these conditions the accurate evaluation of P-glycoprotein-catalyzed doxorubicin transport will be obtained from the Mg2+-sensitive transport into DNA-loaded proteoliposomes.
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PMID:Interaction of doxorubicin with ATP: quantification of complexes and effect on its diffusion into DNA-loaded liposomes--implication for ATP-driven transport studies. 979 32

Previous studies have shown that uptake of the lipophilic opioid, fentanyl, by pulmonary endothelial cells occurs by both passive diffusion and carrier-mediated processes. To evaluate if the latter mechanism also exists in brain endothelium, transport of [3H]fentanyl was examined in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers. Uptake of fentanyl appears to occur via a carrier-mediated process as uptake of [3H]fentanyl by BBMECs was significantly inhibited in a dose-dependent manner by unlabeled fentanyl. Fentanyl uptake was also significantly inhibited by either 4 degrees C or sodium azide/2-deoxyglucose, suggesting that carrier-mediated uptake of fentanyl was an active process. Fentanyl was also tested to determine whether it might be a substrate of the endogenous blood-brain barrier efflux transport system, P-glycoprotein (P-gp). Release of [3H]fentanyl or rhodamine 123, a known substrate of P-gp, previously loaded in the BBMECs was studied in the presence or absence of either fentanyl or verapamil, a known competitive inhibitor of P-gp. Both fentanyl (10 microM) and verapamil (100 microM) decreased release of rhodamine 123 from BBMECs, indicating that fentanyl is a substrate of P-gp in the BBMECs. This was further supported by the observation that uptake of [3H]fentanyl was significantly increased in Mg2+-free medium, a condition known to reduce P-gp activity. However, release of [3H]fentanyl was significantly increased when incubated with either unlabeled fentanyl or verapamil. These results suggest that the active P-gp-mediated extrusion of fentanyl in these cells is overshadowed by an active inward transport process, mediated by an as yet unidentified transporter. In addition, verapamil was shown to be a substrate of both P-gp and the fentanyl uptake transporter.
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PMID:Active transport of fentanyl by the blood-brain barrier. 1021 91


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