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)

The unique yeast translational factor EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA to the ribosome. We have isolated the structural gene encoding EF-3 from the yeast Saccharomyces cerevisiae. The YEF3 gene is found in one copy per haploid genome and is essential for vegetative growth. DNA sequence analysis reveals that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence has two repeats of a nucleotide-binding motif. Each of these repeats shows similarity to the nucleotide-binding motif of hydrophilic, membrane-associated ATPases including human multidrug resistant protein MDR. Factor 3 manifests ribosome-dependent ATP hydrolysis. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity and EF-3 protein levels by 3-5-fold. Yeast strains containing elevated EF-3 protein levels also exhibit increased sensitivity to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. These observations suggest that EF-3 may affect translational accuracy.
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PMID:Isolation and characterization of the structural gene encoding elongation factor 3. 197 86

The renal proximal tubule is a major site of injury in a variety of congenital/metabolic diseases including nephropathic cystinosis, the most commonly known cause of renal Fanconi's syndrome. In this lysosomal storage disease there are defects in proximal tubule function within the first few months of life. While culture of renal tubular cells from the urine of these patients is possible, development of immortalized cell lines would insure large numbers of homogeneous cells for studies of renal epithelial cell morphology and pathophysiology in this disease. To develop immortalized cells, cystinotic and normal proximal tubular cells in culture were exposed to an immortalizing vector, containing pZiptsU19 with the temperature sensitive SV40 T-antigen allele tsA58U19 and a neomycin resistance gene, and neomycin-resistant tubular cells were selected for propagation. Ten clones from cystinotic patients have been developed and characterized. All clones express T-antigen at permissive temperature (33 degrees C). Immortalized cells have an epithelial morphology and grow to form confluent monolayers; doubling times vary from 31 to 86 hours. Cystinotic clones are keratin, MDR P-glycoprotein, and alpha-95 kD brush-border associated protein positive but Tamm-Horsfall protein negative by immunocytochemistry, as are normal proximal tubule cells immortalized with this vector. This is consistent with a proximal tubule origin of the cystinotic clones. The cystine content of the cystinotic cells is 70 to 160 times that of normal renal proximal tubular cells in culture, with most of the cystine sequestered in cell lysosomes, confirming that these cell lines express the storage defect.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Renal proximal tubular epithelium from patients with nephropathic cystinosis: immortalized cell lines as in vitro model systems. 756 23

The aim of the present study was to demonstrate that the modulation of P-glycoprotein (Pgp) ATPase activity by peptides, drugs, and chemosensitizers takes place on a common drug pharmacophore. To this end, a highly emetine-resistant Chinese hamster ovary cell line was established, in which Pgp constituted 18% of plasma membrane protein. Reconstituted proteoliposomes, the Pgp content of which was up to 40%, displayed a basal activity of 2.6 +/- 0.45 micromol of Pi/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 micromol of Pi/min/mg of protein) by valinomycin and various Pgp substrates, whereas, to our surprise, gramicidin D, an established Pgp substrate, was inhibitory. Taking advantage of this novel inhibition of Pgp ATPase activity by gramicidin D, a drug competition assay was devised in which gramicidin D-inhibited Pgp ATPase was coincubated with increasing concentrations of various substrates that stimulate its ATPase activity. Gramicidin D inhibition of Pgp ATPase was reversed by Pgp substrates, including various cytotoxic agents and chemosensitizers. The inhibition of the basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We conclude that the various MDR type substrates and chemosensitizers compete on a common drug binding site present in Pgp.
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PMID:Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity. 862 16

1. P-glycoprotein, a 170-180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents. In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and [3'-keto-Bmt1]-[Val2]-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein. 2. P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay. 3. While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase. No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity. PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity. 4. Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833. 5. The binding of 8-azido-[alpha-32P]-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein. 6. The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.
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PMID:Interaction of cyclosporin derivatives with the ATPase activity of human P-glycoprotein. 931 31

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

Some multidrug-resistant cell lines efflux anticancer drugs but do not overexpress the well-known P-glycoprotein pump or Pgp. A 190 kDa or multidrug-resistant associated protein (MRP) has been identified and described as an MDR mediator. Many studies on cells overexpressing MRP and Pgp, show a concentration of the drug inside cytoplasmic vesicles followed by an exocytotic process. We studied daunorubicin (DNR) subcellular distribution in the presence of an H+-ATPase pump inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) and verapamil (VPL) in two human breast adenocarcinoma MCF7 etoposide-resistant and adriamycin-resistant cell lines, overexpressing respectively MRP (MCF7/VP) and Pgp (MCF7/ADR). Nucleo-cytoplasmic distribution of daunorubicin was carried out using scanning confocal microspectrofluorometry. This technique allows the determination of nuclear accumulation of anthracyclines. Our results show that NBD was able to increase the nuclear accumulation of DNR in MCF7/VP but not in MCF7/ADR cells. Similarly, NBD could reverse DNR resistance in MCF7/VP cells but had no effect on DNR cytotoxicity in MCF7/ADR cells. VPL caused a significant increase in nuclear accumulation of DNR in MCF7/VP and MCF7/ADR cells. Incubation of MCF7/VP and MCF7/ADR cells with VPL, increased the sensitivity of these cells. These data demonstrate clearly that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. In cells overexpressing Pgp, drug efflux probably takes place directly at the membrane level.
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PMID:Role of the vacuolar H+-ATPase in daunorubicin distribution in etoposide-resistant MCF7 cells overexpressing the multidrug-resistance associated protein. 947 14

The mechanism of action of multidrug-resistance reversing activity of torilin was studied. In vitro experiments for the accumulation and efflux of vinblastine clearly indicated that MDR reversing effects of torilin would directly be associated with the increase of the intracellular accumulation of anticancer drugs by blocking the drug efflux. Furthermore, torilin increased the membrane ATPase activity from KB-V1 cells, suggesting that torilin might function by inhibiting drug transport mediated by P-glycoprotein.
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PMID:Mode of action of torilin in multidrug-resistant cancer cell lines. 961 16

The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity. Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif. Within this segment, we identified two sets of residues [ERGA --> DKGT (522-525) and T578C] that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein [Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170]. We wished to gain insight into the molecular basis of this defect. For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris. P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes. P-gp proteoliposomes from P. pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells. Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion. Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity. Photolabeling studies with [125I]iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants. The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.
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PMID:Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity. 963 53

Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11168] we presented evidence for a novel Na+- and Cl--dependent H+ transport process in LR73/hu MDR 1 CHO transfectants that likely explains pHi, volume, and membrane potential changes in eukaryotic cells overexpressing the hu MDR 1 protein. To further explore this process, we have overexpressed human MDR 1 protein in yeast strain 9.3 following a combination of approaches used previously [Kuchler, K., and Thorner, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2302-2306; Ruetz, S., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11588-11592]. Thus, a truncated hu MDR 1 cDNA was cloned behind a tandem array of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters to create the yeast expression vector pFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasma membranes confirmed the overexpression of full length, functional, and properly localized hu MDR 1 protein in independently isolated 9.3/pFF1 colonies. Interestingly, relative valinomycin resistance and growth of the 9.3/hu MDR 1 strains are found to strongly depend on the ionic composition of the growth medium. Atomic absorption reveals significant differences in intracellular K+ for 9.3/hu MDR 1 versus control yeast. Transport assays using [3H]tetraphenylphosphonium ([3H]TPP+) reveal perturbations in membrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pHex. ATPase activity of purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectants constructed previously [Hoffman, M. M., et al. (1996) J. Gen. Physiol. 108, 295-313] was compared. MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeast H+ ATPase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and control strains were analyzed for formation of H+ gradients +/- verapamil. Similar pharmacologic profiles are found for verapamil stimulation of MDR 1 ATPase activity and H+ pumping in 9.3/hu MDR 1 ISOV. In sum, these experiments strongly support the notion that hu MDR 1 catalyzes H+ transport in some fashion and lowers membrane potential in yeast when K+ contributes strongly to that potential. In the accompanying paper [Santai, C. T., Fritz, F., and Roepe, P. D. (1999) Biochemistry 38, XXXX-XXXX] the effects of ion gradients on H+ transport by hu MDR 1 are examined.
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PMID:Evidence for altered ion transport in Saccharomyces cerevisiae overexpressing human MDR 1 protein. 1019 38

In the previous paper we presented a variety of data consistent with significant perturbations in 9.3 yeast plasma membrane ion transport upon overexpression of the hu MDR 1 protein. Thus, in this paper, we compare formation of DeltapH for inside-out yeast plasma membrane vesicles (ISOV) prepared from control 9.3/pVT versus 9.3/hu MDR 1 yeast. Since MDR 1 ATPase activity has a broader, more alkaline pH profile relative to endogenous yeast H+ ATPase activity, we analyzed H+ pumping at pH >/= 8.0 in detail in order to selectively amplify hu MDR 1 contributions to H+ movement over those of the endogenous yeast H+ ATPase. We observed: (1) imposition of a Cl- gradient oriented outside to in enhances acidification for 9.3/pVT ISOV (as expected), but decreases acidification for 9.3/hu MDR 1 ISOV; (2) imposition of a Cl- gradient oriented inside to out decreases acidification for 9.3/pVT ISOV (as expected) but enhances acidification for 9.3/hu MDR 1 ISOV; (3) a Na+ gradient oriented in the same direction as the Cl- gradient amplifies the effects due to hu MDR 1 when both gradients are oriented inside to out, but not outside to in. The data are most easily explained by interesting Na+, Cl-, and ATP-dependent H+ transport mediated by hu MDR 1 protein as previously suggested [Hoffman and Roepe (1997) Biochemistry 36, 11153-11168]. These data may help to resolve a variety of conflicting reports in the literature regarding ion transport mediated by hu MDR 1 and have implications for the physiology of a number of polarized epithelia in which hu MDR 1 is endogenously expressed.
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PMID:Effects of ion gradients on H+ transport mediated by human MDR 1 protein. 1019 39


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