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)

Recovery of cell volume in response to osmotic stress is mediated in part by increases in the Cl- permeability of the plasma membrane. These studies evaluate the hypothesis that ATP release and autocrine stimulation of purinergic (P2) receptors couple increases in cell volume to opening of Cl- channels. In HTC rat hepatoma cells, swelling induced by hypotonic exposure increased membrane Cl- current density to 44.8 +/- 7.1 pA/pF at -80 mV. Both the rate of volume recovery and the increase in Cl- permeability were inhibited in the presence of the ATP hydrolase apyrase (3 units/ml) or by exposure to the P2 receptor blockers suramin and Reactive Blue 2 (10-100 microM). Cell swelling also stimulated release of ATP. Hypotonic exposure increased the concentration of ATP in the effluent of perfused cells by 170 +/- 36 nM in the presence of a nucleotidase inhibitor (P < 0.01). In whole-cell recordings with ATP as the charge carrier, cell swelling increased membrane current density approximately 30-fold to 16.5 +/- 10.4 pA/pF. These findings indicate that increases in cell volume lead to efflux of ATP through opening of a conductive pathway consistent with a channel, and that extracellular ATP is required for recovery from swelling. ATP may function as an autocrine factor that couples increases in cell volume to opening of Cl- channels through stimulation of P2 receptors.
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PMID:Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation. 887 55

Plasma membrane P-glycoprotein is known as an ATP-dependent drug efflux pump that confers multidrug resistance to tumor cells. None of the reported purification procedures worked properly for our P-glycoprotein-overproducing cell lines, i.e. murine lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/COL10, and human lymphoblastic leukemia CEM/VLB5 cells. We have thus developed a general procedure for efficient purification of P-glycoprotein by combining solubilization with sodium dodecyl sulfate and chromatography on ceramic hydroxyapatite. This procedure was successful for the three cell lines and yielded 70% of the P-glycoprotein present in the starting plasma membranes with more than 99% purity. After exchanging sodium dodecyl sulfate into dodecyl maltoside and reconstitution into liposomes, purified P-glycoprotein exhibited a specific ATPase activity of about 200 nmol/min/mg, which was very similar to that obtained for P-glycoprotein solubilized and purified with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. This ATPase activity was sensitive to orthovanadate inhibition and stimulated by verapamil and other drugs. More importantly, drug transport properties of the reconstituted P-glycoprotein were comparable with those of P-glycoprotein embedded in plasma membranes. Since it is virtually devoid of lipids, this preparation is suitable for both functional and structural investigations.
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PMID:Efficient purification and reconstitution of P-glycoprotein for functional and structural studies. 891 May 34

L-Asparagine stimulates bi-directional Ca(2+) flows and induces ornithine decarboxylase in Reuber H-35 hepatoma cells. Previously it has been shown that these effects are completely, but reversibly inhibited by lanthanum chloride. In this study we examined the role(s) of Ca(2+) flows using more specific Ca(2+) flow inhibitors. It was shown that ornithine decarboxylase induction was inhibited by CdCl(2) and verapamil at concentrations above 1 mu M and 100 mu M respectively, but was unaffected by as much as 300 mu M NiCl(2), 1 mM nifedipine, or 10 mu M omega-conotoxin. Enzyme induction was blocked by the Ca(2+)-ATPase pump antagonists vanadate and Compound 48/80 in a dose-dependent manner. These results, taken together with the observations that extracellular Ca(2+) is essential for enzyme induction but a substantial elevation of cytoplasmic [Ca(2+)] is not, suggest that Ca(2+) inflow independent of the receptor-activated Ca(2+) channels, and the Ca(2+)-ATPase mediated Ca(2+) out-flow, are both important factors in the action of L-asparagine.
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PMID:Characterization of Ca(2+) flows essential in ornithine decarboxylase induction by L-asparagine in rat hepatoma cells using Ca(2+) flow inhibitors. 913 51

Cancer cells, despite growing aerobically, have the propension to utilize the glycolytic pathway as energy source. This biochemical phenotype is accompanied by a decreased content of mitochondria and, paradoxically, by enhanced transcription of nuclear and mitochondrial-encoded genes for the enzymes of oxidative phosphorylation (OXPHOS). The role of OXPHOS enzymes in normal and neoplastic cell growth has been studied in liver regeneration and human hepatocellular carcinoma. In early liver regeneration characterized by active mtDNA replication, a decrease in the content and activity of ATP synthase occurs while transcription of the ATPsyn beta nuclear gene is activated. Translation of ATP synthase subunits seems, on the contrary, to be less effective in this phase. In the second replicative phase of liver regeneration, the repression of ATPsyn beta translation is relieved and normal cell growth starts. In this replicative phase the recovery of the liver mass appears to be directly related to the recovery of the OXPHOS capacity. Mitochondria isolated from biopsies of human hepatocellular carcinoma exhibit a decreased rate of respiratory ATP synthesis (OXPHOS) and a decreased ATPase activity. The decline in the activity of the ATP synthase is found to be associated with a decreased content of the ATPsyn beta in the inner mitochondrial membrane. In neoplastic tissue the ATPase inhibitor protein (IF1) is overexpressed. This could contribute to prevent hydrolysis of glycolytic ATP in cancer cells. A peptide segment of IF1 (IF1-(42-58)-peptide), constructed by chemical synthesis, proved to be equally effective as IF1 in inhibiting the ATPase activity of the ATP synthase complex in the mitochondrial membrane deprived of IF1. The synthetic peptide might turn out to be a useful tool to develop immunological approaches for the control of neoplastic growth.
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PMID:Oxidative phosphorylation enzymes in normal and neoplastic cell growth. 938 98

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

Composition and amount of 45Ca2+-binding proteins in the inner membrane fraction of rat liver and Zajdela hepatoma mitochondria were determined. In the inner membrane of liver mitochondria, three major 45Ca2+-binding polypeptides: a protein of approximately 130 kDa (carbamoyl-phosphate synthetase), a glycoprotein of 43-44 kDa (previously considered as the calcium uniporter), and 29-30 kDa protein were found. These components were absent (130 kDa component) or relatively reduced (43-44 kDa and 29-30 kDa components) in the inner membrane of hepatoma mitochondria. Previously unknown low molecular mass polypeptides, having very high Ca2+-binding ability, were found in the inner membrane of hepatoma mitochondria. One of them might be the natural Ca2+-binding inhibitor of H+-ATPase.
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PMID:Calcium binding to polypeptides of rat liver and Zajdela hepatoma mitochondrial inner membranes. 950 39

We studied the effects of bafilomycin A1, a potent and specific inhibitor of vacuolar H+ ATPase (V-ATPase), on the process of autophagy in rat hepatoma cell line, H-4-II-E cells. To induce autophagy, cells were transferred from Dulbecco's modified Eagle medium containing 12% fetal calf serum into Hanks' balanced salt solution. When bafilomycin A1 was added to Hanks' balanced salt solution, endogenous protein degradation was strongly inhibited and numerous autophagosomes accumulated in H-4-II-E cells, whereas autolysosomes decreased in number. Acid phosphatase activity was not detected in the autophagosomes which accumulated in the presence of bafilomycin A1, suggesting that fusion between autophagosomes and lysosomes was disturbed by this drug. Inhibition of the fusion was reversible, and the autophagosomes changed into autolysosomes after the removal of the inhibitor. Bafilomycin A1 also prevented the appearance of endocytosed HRP in autophagic vacuoles. These results suggested that acidification of the lumenal space of autophagosomes or lysosomes by V-ATPase is important for the fusion between autophagosomes and lysosomes.
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PMID:Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. 963 28

The polarized rat hepatoma/human fibroblast hybrid cell line, WIF-B, forms apical vacuoles into which cholephilic substances are secreted. We studied expression, localization, and function of the apical conjugate export pump, Mrp2, in WIF-B cells. Mrp2, the apical isoform of the multidrug resistance protein, alternatively termed canalicular Mrp (cMrp) or canalicular multispecific organic anion transporter (cMoat), is a 190-kd membrane glycoprotein mediating adenosine triphosphate (ATP)-dependent transport of glucuronides, glutathione S-conjugates, and other amphiphilic anions across the hepatocyte canalicular membrane into bile. Expression of the rat mrp2 gene in WIF-B cells was shown by reverse-transcription polymerase chain reaction (PCR), followed by sequencing of the amplified 789-bp fragment. Immunoblotting, using antibodies reacting with the amino-terminal or with the carboxyl-terminal sequence of rat Mrp2, detected the 190-kd glycoprotein in WIF-B cell homogenates. Immunofluorescence microscopy localized Mrp2 to the apical membrane domain. Preloading of WIF-B cells with a membrane-permeable ester of the calcium-dependent fluorescent indicator, Fluo-3, was followed by Mrp2-mediated secretion of the amphiphilic anion, Fluo-3, into the apical vacuoles. This transport was potently inhibited by cyclosporin A added to the culture medium. Direct measurements of ATP-dependent transport into Mrp2-containing plasma membrane vesicles in comparison with Mrp2-deficient vesicles established that Fluo-3 is transported by Mrp2 with a Km value of 3.7 micromol/L. Our results indicate that the polarized WIF-B cells express the rat ortholog of the apical conjugate-transporting ATPase, Mrp2. The function of Mrp2 as well as the action of inhibitors can thus be analyzed by use of the fluorescent amphiphilic anion, Fluo-3.
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PMID:Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF-B. 979 19

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.
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PMID:In vitro and in vivo reversal of cancer cell multidrug resistance by the semi-synthetic antibiotic tiamulin. 980 34

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein's interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.
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PMID:Hepatitis C virus core protein interacts with cellular putative RNA helicase. 1007 32


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