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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Considerable uncertainty surrounds the stoichiometry of coupling of ATP hydrolysis to drug pumping by P-glycoprotein, the multidrug transporter. To estimate relative turnovers for pumping of the drug vinblastine and ATP hydrolysis, we began by measuring the number of P-glycoprotein molecules on the surface of murine NIH3T3 cells expressing the human MDR1 gene. Fluorescence of cells treated with monoclonal antibody UIC2 was determined as a function of (i) amount of antibody at a fixed number of cells and (ii) increasing cell number at constant antibody. The two together gives 1.95 x 10(6) P-glycoprotein molecules/cell. Initial uptake rates of vinblastine +/- verapamil measure the ability of P-glycoprotein to extract vinblastine from the plasma membrane before it enters the cell. As a function of [vinblastine] at 37 degrees C, they give the maximum rate of this component of outward pumping as 2.1 x 10(6) molecules s-1 cell-1 or a turnover number of 1.1 s-1. Initial rates of one-way efflux as a function of [vinblastine] at 25 degrees C +/- glucose give the maximum rate of this component of pumping as 0.59 x 10(6) molecules s-1 cell-1. The ratio of ATPase activity of P-glycoprotein at 37 and 25 degrees C is 4.6. Appropriating this ratio for pumping, maximum one-way efflux at 37 degrees C is 4.6 x 0.59 = 2.7 x 10(6) molecules s-1 cell-1, a turnover number of 1.4 s-1. The vinblastine-stimulated ATPase activity of P-glycoprotein has a turnover number of 3.5 s-1 at 37 degrees C, giving 2.8 molecules of ATP hydrolyzed for every vinblastine molecule transported in a particular direction. These calculations involve several approximations, but turnover numbers for pumping of vinblastine and for vinblastine-stimulated ATP hydrolysis are comparable. Thus, ATP hydrolysis is probably directly linked to drug transport by P-glycoprotein.
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PMID:Relation between the turnover number for vinblastine transport and for vinblastine-stimulated ATP hydrolysis by human P-glycoprotein. 926 Nov 21

In the human multidrug transporter (MDR1), three serine residues located in the "linker" region of the protein are targets of in vivo phosphorylation. These three serines, or all eight serines and threonines in the linker, were substituted by alanines (mutants 3A and 8A) or with glutamic acids (mutants 3E and 8E). The wild-type and mutant proteins were expressed in baculovirus-infected Spodoptera frugiperda (Sf9) ovarian insect cells, and the vanadate-sensitive, drug-stimulated ATPase activity was measured in isolated membrane preparations. The maximum drug-stimulated MDR1-ATPase activity was similar for the wild-type and the mutant proteins. However, wild-type MDR1, which is known to be phosphorylated in Sf9 membranes, and the 3E and 8E mutants, which mimic the charge of phosphorylation, achieved half-maximum activation of MDR1-ATPase activity at lower verapamil, vinblastine, or rhodamine 123 concentrations than the nonphosphorylatable 3A and 8A variants. For some other drugs (e.g. valinomycin or calcein acetoxymethylester) activation of the MDR1-ATPase for any of the mutants was indistinguishable from that of the wild-type protein. Kinetic analysis of the data obtained for the 3A and 8A MDR1 variants indicated the presence of more than one drug interaction site, exhibiting an apparent negative cooperativity. This phenomenon was not observed for the wild-type or the 3E and 8E MDR1 proteins. The dependence of the MDR1-ATPase activity on ATP concentration was identical in the wild-type and the mutant proteins, and Hill plots indicated the presence of more than one functional ATP-binding site. These results suggest that phosphorylation of the linker region modulates the interaction of certain drugs with MDR1, especially at low concentrations, although phosphorylation does not alter the maximum level of MDR1-ATPase activity or its dependence on ATP concentration.
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PMID:Phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated ATPase activity. 928 20

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.
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PMID:P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity. 937 74

The transmembrane distribution of phospholipids in the membranes of eukaryotic cells depends on specific proteins (called flippases). The aminophospholipid translocase is responsible for the sequestration of phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet of plasma membranes. Several laboratories are presently working on the identification, purification and cloning of this Mg-ATPase, first recognized in the human red cell membrane. In accordance with the 1992 hypothesis of Higgins and Gottesman, proteins of the MDR1 family appear to be able to translocate certain phospholipids from the inner to the outer monolayer of the plasma membrane. It has been reported in particular that expression of the human MDR3 and mouse mdr2 genes promote translocation of long chain phosphatidylcholine, while expression of the MDR1 gene stimulates the outward motion of phospholipids possessing at least one short chain. ATP-independent flippases activities were recognized not only in microsomes but also in Golgi membranes.
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PMID:Aminophospholipid translocase and proteins involved in transmembrane phospholipid traffic. 946 21

The FDA approved HIV-1 protease inhibitors, ritonavir, saquinavir, and indinavir, are very effective in inhibiting HIV-1 replication, but their long-term efficacy is unknown. Since in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether these protease inhibitors are recognized by the MDR1 multidrug transporter (P-glycoprotein, or P-gp), thereby reducing their intracellular accumulation. In vitro studies in isolated membrane preparations from insect cells infected with MDR1-expressing recombinant baculovirus showed that these inhibitors significantly stimulated P-gp-specific ATPase activity and that this stimulation was inhibited by SDZ PSC 833, a potent inhibitor of P-gp. Furthermore, photoaffinity labeling of P-gp with the substrate analogue [125I]iodoarylazidoprazosin (IAAP) was inhibited by all three inhibitors. Cell-based approaches to evaluate the ability of these protease inhibitors to compete for transport of known P-gp substrates showed that all three HIV-1 protease inhibitors were capable of inhibiting the transport of some of the known P-gp substrates but their effects were generally weaker than other documented P-gp modulators such as verapamil or cyclosporin A. Inhibition of HIV-1 replication by all three protease inhibitors was reduced but could be restored by MDR1 inhibitors in cells expressing MDR1. These results indicate that the HIV-1 protease inhibitors are substrates of the human multidrug transporter, suggesting that cells in patients that express the MDR1 transporter will be relatively resistant to the anti-viral effects of the HIV-1 protease inhibitors, and that absorption, excretion, and distribution of these inhibitors in the body may be affected by the multidrug transporter.
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PMID:HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. 953 Feb 86

The human multidrug transporter (MDR1 or P-glycoprotein) is an ATP-dependent cellular drug extrusion pump, and its function involves a drug-stimulated, vanadate-inhibited ATPase activity. In the presence of vanadate and MgATP, a nucleotide (ADP) is trapped in MDR1, which alters the drug binding properties of the protein. Here, we demonstrate that the rate of vanadate-dependent nucleotide trapping by MDR1 is significantly stimulated by the transported drug substrates in a concentration-dependent manner closely resembling the drug stimulation of MDR1-ATPase. Non-MDR1 substrates do not modulate, whereas N-ethylmaleimide, a covalent inhibitor of the ATPase activity, eliminates vanadate-dependent nucleotide trapping. A deletion in MDR1 (Delta amino acids 78-97), which alters the substrate stimulation of its ATPase activity, similarly alters the drug dependence of nucleotide trapping. MDR1 variants with mutations of key lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which bind but do not hydrolyze ATP, do not show nucleotide trapping either with or without the transported drug substrates. These data indicate that vanadate-dependent nucleotide trapping reflects a drug-stimulated partial reaction of ATP hydrolysis by MDR1, which involves the cooperation of the two nucleotide binding domains. The analysis of this drug-dependent partial reaction may significantly help to characterize the substrate recognition and the ATP-dependent transport mechanism of the MDR1 pump protein.
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PMID:Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1. Cooperation of the nucleotide binding domains. 955 60

Twenty-one 2-chloro-N10-substituted phenoxazines have been synthesized and characterized as potential modulators of multidrug resistance (MDR). Many of the compounds, at a concentration of 100 microM, enhanced accumulation of vinblastine (VLB) in drug-resistant KB8-5 cells to a greater extent than the same concentration of verapamil (VRP). However, the effects on VLB accumulation were specific, because these derivatives had little activity in the parental drug-sensitive line KB3-1. The compounds slowed the efflux of VLB from KB8-5 cells, suggesting that the chlorophenoxazines, like VRP, can inhibit P-glycoprotein (P-gp)-mediated efflux of VLB from this cell line. Two of the chlorophenoxazine derivatives, and also VRP, were able to stimulate the vanadate-sensitive ATPase activity attributable to P-gp in membranes isolated from MDR1 baculovirus-infected Sf9 cells. This result suggests that these modulators exert their effect by directly interacting with P-gp. Apart from the parent unsubstituted molecule, 2-chlorophenoxazine, there was a good correlation between log10P and the ability of the compounds to enhance VLB accumulation in KB8-5. This suggests that lipophilicity of a modulator is important, but is not the sole determinant of potency. Within this series of compounds, the optimal structural features for MDR modulation include a hydrophobic phenoxazine ring with a -Cl atom in the C-2 position and a tertiary amine group four carbons from the tricyclic ring. Many of the agents at the IC10 concentration completely reversed the 37-fold VLB resistance in KB8-5 cells. The most active agents in KB8-5 were able to partially reverse VLB resistance in an MDR colon carcinoma cell line GC3/c1 and completely reversed the 86-fold VLB resistance in the MDR1-overexpressing breast carcinoma cell line BC19/3. These same agents could only partially sensitize BC19/3 cells to taxol and doxorubicin, suggesting that the chlorophenoxazine derivatives show some specificity for modulating VLB resistance.
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PMID:Characterization of 2-chloro-N10-substituted phenoxazines for reversing multidrug resistance in cancer cells. 961 55

One major form of multiple drug resistance (MDR) to cancer therapeutic agents is mediated by overexpression of P-glycoprotein, a membrane ATPase that serves as a drug efflux pump. In humans, this protein is the product of the MDR1 gene. We have used chemically modified antisense oligonucleotides to reduce expression of P-glycoprotein in multidrug-resistant fibroblasts and colon carcinoma cells. Although several types of oligonucleotides were tested, compounds having a phosphorothioate backbone and a methoxyethoxy (ME) group at the 2' position of the ribose ring proved to have the greatest potency. Thus, phosphorothioate 2'-ME oligonucleotides directed against either the AUG codon region or the stop codon region of the MDR1 message produced substantial (50-70%) inhibition of P-glycoprotein expression at concentrations of < or = 50 nM. In addition, such treatment resulted in augmented drug uptake as measured by flow cytometry. Unmodified phosphorothioate compounds of the same sequence were active only in the micromolar range. We also tested the ability of several potential delivery agents to enhance the pharmacological effectiveness of anti-MDR1 oligonucleotides. Both commercial Lipofectin, a well known liposomal transfection agent, and a liposomal preparation based on a novel "facial amphiphile" were effective in enhancing their pharmacological effects of antisense oligonucleotides. A Starburst dendrimer, a type of cationic polymer, was also effective in oligonucleotide delivery. This report emphasizes that significant improvements in antisense pharmacology can be made through judicious use of both chemical modifications of oligonucleotides and appropriate delivery systems.
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PMID:Novel chemically modified oligonucleotides provide potent inhibition of P-glycoprotein expression. 965 87

Human Pgp from the vinblastine-resistant cell line, KB-V1, can be purified by sequential conventional chromatography on DEAE-sepharose CL-6B resin followed by a wheat germ agglutinin column. By including glycerol (osmolyte protectant) and lipid during the solubilization and chromatography procedures most of the biological activity of Pgp can be retained. The activity of Pgp in the detergent extract or in the concentrated column fractions is stable for at least 8-10 months when stored at -80 degrees. However, repeated cycles of freezing and thawing of fractions result in considerable loss of activity. We have purified Pgp from KB-C1 (a subclone of KB 3-1 that is resistant to 1 microgram/ml colchicine) by following the same protocol. When this method was used for purification of Pgp from MDR1-transfected NIH 3T3 transfectants (N3-V2400, grown in the presence of 2.4 micrograms/ml vinblastine), the protein was eluted with 0.1 M NaCl from the DEAE-Sepharose CL-6B column as usual. However, during WGA lectin chromatography, the protein was eluted with a lower concentration of sugar (0.1 M instead of 0.25 M NAG). This altered elution pattern appears to be due to a difference in the glycosylation of human Pgp in mouse NIH 3T3 cells. This is consistent with the observation that human Pgp expressed in NIH 3T3 cells migrates faster compared to the protein from KB-V1 cells on 8-10% acrylamide gel. Similarly, other workers have purified Chinese hamster Pgp either by a single-step chromatography on Reactive Red 120 agarose or by a combination of anion exchange and immunoaffinity chromatography (see the article by Senior et al. for the purification and properties of ATPase activity of Chinese hamster Pgp). The high level of drug-stimulated ATP hydrolysis by Pgp (Table I), like other ion-transporting ATPases, indicates that this is a high-capacity pump that can function as an effective multidrug transporter. This is further supported by the qualitative demonstration of ATP-dependent vinblastine transport in proteoliposomes reconstituted with pure Pgp (see Fig. 2). Thus, these experiments provide strong evidence that purified Pgp retains its activity and that it functions as an ATP-dependent drug transporter.
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PMID:Purification and reconstitution of human P-glycoprotein. 971 77

MDR1, an ABC transporter that confers multidrug resistance in tumor cells, is constitutively expressed in normal liver canalicular membrane. Human MDR1-expressing multidrug-resistant cells display increased resistance to estradiol-17beta(beta-D-glucuronide) (E217G). MDR1 substrates/modulators inhibit adenosine triphosphate (ATP)-dependent transport of E217G in the rat canalicular membrane and protect against E217G-mediated cholestasis in isolated perfused rat liver. The present studies were designed to determine if E217G is a substrate for MDR1 using a baculovirus expression system and if other estrogen glucuronides interact with MDR1. ATP-dependent transport of E217G (10 micromol/L) was linear for up to 2 minutes and yielded a rate of 45.6 pmol/min/mg protein in membrane vesicles from Sf9 cells infected with MDR1-baculovirus. This transport was saturable (Km = 62 micromol/L) and occurred into an osmotically sensitive space. ATP-dependent transport of E217G (10 micromol/L) was inhibited 63% by 10 micromol/L daunomycin, but not by 100 micromol/L S-(2,4-dinitrophenyl)glutathione (GS-DNP) (a substrate for canalicular multispecific organic anion transporter [cMOAT]). Glucuronide conjugates of the estrogen D-ring (100 micromol/L), estriol-17beta(beta-D-glucuronide) (E317G) and estriol-16(beta-D-glucuronide) (E316G), inhibited MDR1-mediated E217G transport by 58% and 35%, respectively. In contrast, noncholestatic glucuronides, estradiol-3-(beta-D-glucuronide) (E23G) or estradiol-3-sulfate-17beta(beta-D-glucuronide) (E23SO417G), had no effect. E217G neither stimulated MDR1 ATPase activity nor inhibited verapamil-stimulated ATPase activity. Infusion of 1.5 micromol/L doxorubicin or 1 micromol/L taxol protected against cholestasis induced by E316G and E317G in isolated perfused rat liver. These studies identify E217G, and probably E316G and E317G, as endogenous substrates for MDR1.
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PMID:Adenosine triphosphate-dependent transport of estradiol-17beta(beta-D-glucuronide) in membrane vesicles by MDR1 expressed in insect cells. 979 24


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