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)

We expressed P-glycoproteins (P-gps) encoded by the three mouse mdr genes in the membranes of secretory vesicles (SV) accumulating in the yeast mutant strain sec 6-4. Expression of the Mdr1 and Mdr3 isoforms in SV membranes caused a significant increased accumulation of the drug vinblastine (VBL) over background levels measured in control SV. The Mdr1/Mdr3-mediated increased drug accumulation could be completely abolished by the P-gp modulator verapamil. By contrast, overexpression of Mdr2 in these vesicles failed to increase intravesicular VBL accumulation over background levels. Mdr3-mediated VBL transport was not affected by changes in the membrane potential, since identical rates of VBL uptake were measured in the presence or absence of the endogenous proton-translocating PMA1 H(+)-ATPase responsible for the strong electrochemical membrane potential across SV membranes. Moreover, in the presence of a delta micro-H+ across the SV membranes (inside positive) of almost 90 mV, we detected in Mdr3-expressing SV an enhanced accumulation of the lipophilic cation and P-gp substrate tetraphenylphosphonium, suggesting that P-gp-mediated uptake of this cation occurs against an intravesicular depolarized membrane. Likewise, VBL transport in Mdr3-expressing SV was not affected by the presence or absence of a steep proton gradient (inside acid) and was independent of any proton movements, excluding a proton synport or antiport mechanism for P-gp-mediated drug transport. Finally, we could demonstrate that colchicine accumulation in Mdr3-expressing SV occurred against a significant substrate concentration gradient, reaching a 7-fold increase in intravesicular colchicine concentration above the extravesicular medium drug concentration. Our studies show that SV isolated from the temperature-sensitive yeast sec 6-4 mutants are an ideal tool to express and to functionally characterize heterologous membrane proteins, in general and P-gps, in particular.
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PMID:Functional expression of P-glycoproteins in secretory vesicles. 790 18

P-glycoproteins (P-gps) encoded by the mouse mdr2 and mdr3 genes were expressed in secretory vesicles (SVs) from the yeast mutant sec6-4, and their capacity to function as a lipid translocase/flippase was tested. An assay that uses a fluorescent phosphatidylcholine (PC) analog was developed to quantitate asymmetric lipid distribution in the outer and inner leaflets of the lipid bilayer of these vesicles. Mdr2 expression in SVs caused a time- and temperature-dependent enhancement of PC translocation to the inner leaflet of the membrane. The Mdr2-mediated effect was specific since expression of Mdr3 in these vesicles was without effect on the membrane distribution of PC. Increased Mdr2-mediated PC translocation was strictly ATP and Mg2+ dependent, was abrogated by the ATPase inhibitor vanadate and the P-gp modulator verapamil, but was insensitive to the presence of excess of the multidrug resistance drugs colchicine and vinblastine.
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PMID:Phosphatidylcholine translocase: a physiological role for the mdr2 gene. 791 58

The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase II clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound. Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEM/VLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/val185) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase II gene, no cross-resistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRF/VCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCl. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.
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PMID:Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440). 932 44

P-gp (Pgp) is a cell surface ATPase which confers resistance to many of the most active chemotherapy drugs, including taxol, doxorubicin, and vinca alkaloids. Pgp can be detected in human cancers by immunohistochemistry, RNA probes, or by functional assays utilizing transported fluorescent dyes such as rhodamine. The expression of Pgp in untreated human cancers is highly variable, being almost universal in colon, hepatocellular carcinoma, and renal cell cancers, less common in breast, ovarian, and lymphoid malignancies. At least part of the heterogeneity is attributable to different definitions of positivity even with a given method of detection. In chemotherapy naive cancers, resistant cells may not occur very frequently. Whilst the Goldie-Coldman hypothesis predicts treatment failure if 1 cell in 10(6) expresses a resistance mechanism, no method of detection yet described can reliably achieve this. The field has reached a stage in which it may be possible to detect Pgp accurately in advanced cancers which have failed chemotherapy allowing phase II clinical trials to be performed in Pgp-positive tumors. In terms of which Pgp inhibitors are selected for clinical study it is likely that selection of Pgp inhibitors with nM potency to bind to Pgp will be important. Such drugs should undergo extensive phase I trial evaluation to assess pharmacokinetic interactions with a range of cytotoxic drugs before entering randomized trials. In randomized clinical trials Pgp detection may be less important, as disease-free survival and overall survival would be the key end-points, but the Pgp positivity of relapsed disease would indicate if treatment with inhibitors of Pgp-eliminated Pgp-expressing clones. The accurate detection of Pgp in human cancers is being refined and will be an essential component of future Pgp inhibitor clinical trials. Finally, these trails must be of sufficient size (> 500 patients per arm) to reliably detect clinically meaningful differences.
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PMID:Testing the role of P-glycoprotein expression in clinical trials: applying pharmacological principles and best methods for detection together with good clinical trials methodology. 947 46

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 inhibition of the Na+/K+-ATPase by cardiotonic drugs like ouabain deeply perturbs both the properties of the cell membrane and the ionic composition of the cytoplasm and hence alters fundamental cell reactions. These three types of reactions may be involved in the stimulation of multidrug resistance 1 (MDR-1) gene expression and the synthesis of permeability glycoprotein [P-glycoprotein (P-gp)]. We have determined whether ouabain, which binds to an extracellular motif of the Na+/K+-ATPase, stimulates MDR-1 gene expression by measuring both mRNA and protein and whether the resulting P-gp extrudes hydrophobic compounds and causes resistance to antimitotic agents. The experiments were performed on Calu-3 cells, a human cell line from a pulmonary carcinoma. Northern blotting showed that treating the cells with submicromolar concentrations of ouabain stimulated MDR-1 gene expression within 24 h. The ouabain-induced stimulation of MDR-1 expression was not restricted to Calu-3 cells but also occurred in human carcinomatous colon (T-84 and HT-29) and hepatic (H7V3) cells. However, it is not ubiquitous because it was not found in HeLa cells. The stimulation was reproduced by other Na+/K+-ATPase inhibitors and occurred via enhanced gene transcription, apparently due to the increased cytosolic calcium concentration. Ouabain also increased the membrane content of P-gp, as detected by immunoblotting and immunohistology. We have developed a microvideo assay based on the properties of acetoxymethyl ester calcein and calcein to show that this P-gp extruded the hydrophobic acetoxymethyl ester calcein. Ouabain also caused the Calu-3 cells to become resistant to doxorubicin and vinblastine. Thus, although ouabain acts extracellularly, it may stimulate MDR-1 gene expression and P-gp synthesis and make cells resistant to hydrophobic cytotoxic compounds.
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PMID:Drug resistance induced by ouabain via the stimulation of MDR1 gene expression in human carcinomatous pulmonary cells. 1124 85

P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.
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PMID:Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures. 1155 29

1. Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P-glycoprotein. Wild-type (wt) and the mutant (cys-less) forms of P-gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies. 2. No significant differences were observed in substrate ([(3)H]-azidopine) binding to wt or cys-less P-gp. Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace [(3)H]-azidopine from the wt or cys-less P-gp. These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions. 3. The Michaelis-Menten characteristics of basal ATP hydrolysis of the two isoforms of P-gp were identical. The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the K(m) was unchanged in cys-less P-gp compared to control. However, cys-less P-gp displayed lower overall maximal ATPase activity (62%), a decreased K(m) and a lower degree of stimulation (76%) in the presence of the modulator nicardipine. 4. Therefore, the serine to cysteine mutations in P-gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways. The wt and cys-less P-gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys-less P-gp as a template for future cysteine-directed structure/function analysis.
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PMID:Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein. 1173 36

The human multidrug resistance-associated protein(MRP1) is an ATP-dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well-characterized multidrug transporter P-gp, MRP1 comprises two nucleotide-binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P-gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione-S-transferase (GST) or the maltose-binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST-fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent K(m) of 340 microm and a V(max) of 6.0 nmol P(I) x mg-1 x min-1, and a K(m) of 910 microm ATP and a V(max) of 7.5 nmol P(I) x mg-1 x min-1, respectively. Remarkably, S-decyl-glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S-decyl-glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations.
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PMID:S-decyl-glutathione nonspecifically stimulates the ATPase activity of the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 (ABCC1). 1213 86

The human multidrug resistance P-glycoprotein (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) family of transport proteins, actively transports many cytotoxic compounds out of the cell. ABC transporters have two nucleotide-binding domains (NBD) and two transmembrane domains. The presence of the conserved "signature" sequence (LSGGQ) in each NBD is a unique feature in these transporters. The function of the signature sequences is unknown. In this study, we tested whether the signature sequences ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) in P-gp are in close proximity to the opposing Walker A consensus nucleotide-binding sequences ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1). Pairs of cysteines were introduced into a Cys-less P-gp at the signature and "Walker A" sites and the mutant P-gps were subjected to oxidative cross-linking. At 4 degrees C, when thermal motion is low, P-gp mutants (L531C(Signature)/C1074(Walker A) and C431(Walker A)/L1176C(Signature) were cross-linked. Cross-linking inhibited the drug-stimulated ATPase activities of these two mutants. Their activities were restored, however, after addition of the reducing agent, dithiothreitol. Vanadate trapping of nucleotide at the ATP-binding sites prevented cross-linking of the mutants. These results indicate that the signature sequences are adjacent to the opposing Walker A site. They likely participate in forming the ATP-binding sites and are displaced upon ATP hydrolysis. The resulting conformational change may be the signal responsible for coupling ATP hydrolysis to drug transport by inducing conformational changes in the transmembrane segments.
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PMID:The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence. 1222 74


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