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)

Approximately 50% of Helicobacter pylori strains produce a toxin in vitro that induces vacuolation of eukaryotic cells. To determine whether ion transport pathways are important in the formation of toxin-induced vacuoles, HeLa cells were incubated with H. pylori toxin in the presence of nine different inhibitors of ion-transporting ATPases. Oligomycin, an inhibitor of predominantly F1F0-type ATPases, had no effect on toxin activity. Inhibitors of predominantly V-type ATPases, exemplified by bafilomycin A1, inhibited the formation of vacuoles in response to the H. pylori toxin and reversed the vacuolation induced by the toxin. In contrast, at concentrations of > or = 100 nM, ouabain and digoxin, inhibitors of the Na(+)-K+ ATPase, potentiated the activity of H. pylori toxin. The inhibitory effects of bafilomycin A1 could not be overcome by the potentiating effects of ouabain. These data suggest that intact activity of the vacuolar ATPase of eukaryotic cells is a critical requirement in the pathogenesis of cell vacuolation induced by H. pylori toxin and that vacuole formation by this toxin is associated with altered cation transport within eukaryotic cells.
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PMID:Effects of ATPase inhibitors on the response of HeLa cells to Helicobacter pylori vacuolating toxin. 845 46

Although the majority of known vacuolar proteins transit through the secretory pathway, two vacuole-resident proteins have been identified that reach this organelle by an alternate pathway. These polypeptides are targeted to the vacuole directly from the cytoplasm by a novel import mechanism. The best characterized protein that uses this pathway is aminopeptidase I (API). API is synthesized as a cytoplasmic precursor containing an amino-terminal propeptide that is cleaved off when the protein reaches the vacuole. To dissect the biochemistry of this pathway, we have reconstituted the targeting of API in vitro in a permeabilized cell system. Based on several criteria, the in vitro import assay faithfully reconstitutes the in vivo reaction. After incubation under import conditions, API is processed by a vacuolar-resident protease, copurifies with a vacuole-enriched fraction, and becomes inaccessible to the cytoplasm. These observations demonstrate that API has passed from the cytoplasm to the vacuole. The reconstituted import process is dependent on time, temperature, and energy. ATP gamma S inhibits this reaction, indicating that API transport is ATP driven. API import is also inhibited by GTP gamma S, suggesting that this process may be mediated by a GTP-binding protein. In addition, in vitro import requires a functional vacuolar ATPase; import is inhibited both in the presence of the specific V-ATPase inhibitor bafilomycin A1, and in a yeast strain in which one of the genes encoding a V-ATPase subunit has been disrupted.
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PMID:In vitro reconstitution of cytoplasm to vacuole protein targeting in yeast. 855 40

Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATP-driven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes. H+ pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N'-dicyclohexylcarbodiimide. In addition, the epicotyl H+ pumping activity was inactivated by oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate ( > or = 100 mM) was correlated with the release of V1 complexes from the membrane. In contrast, H+ pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the V-ATPase inhibitors, and resistant to oxidation. Unlike the epicotyl inhibitors, and resistant to oxidation. Unlike the epicotyl H(+)-ATPase, the fruit H(+)-ATPase activity was partially inhibited by 200 microM vanadate. Cold inactivation treatment failed to inhibit H+ pumping activity of the fruit membranes, even though immunoblasts showed that V1 complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 microM vanadate from 30% to 60%. These results suggest the presence of two H(+)-ATPases in the fruit preparation: a V-ATPase and an unidentified vanadate-sensitive H(+)-ATPase. Attempts to separate the two activities in their native membranes on linear sucrose density density gradients were unsuccessful. However, following detergent-solubilization and centrifugation on a glycerol density gradient, the two ATPase activities were resolved: a nitrate-sensitive V-type ATPase that is also partially inhibited by 200 microM vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.
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PMID:On the mechanism of hyperacidification in lemon. Comparison of the vacuolar H(+)-ATPase activities of fruits and epicotyls. 856 39

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H(+)-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement of affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites of organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the facuolar H(+)-ATPase.
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PMID:Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase. 871 68

A 1084 base pair partial cDNA showing similarity to the C subunit of the vacuolar ATPase (V-ATPase) was isolated on a clone from a cDNA library made from salivary glands from 3-day-old feeding adult Amblyomma americanum (L.) female ticks. The 5' end was completed using primer extension and the two pieces joined to form a complete cDNA of 1373 bp. This mRNA is expressed in embryos and the salivary glands of unfed adults and adult females at all stages of feeding. Specific inhibitors of the V-ATPase decrease the rate of dopamine-stimulated secretion of isolated salivary glands, but not as much as ouabain, an inhibitor of the Na+, K+ ATPase, indicating that a V-ATPase may participate in the mechanism of salivary fluid secretion in A. americanum, but the volume of saliva secreted is more dependent on an active Na+, K+ ATPase.
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PMID:Cloning and sequence of a gene for a homologue of the C subunit of the V-ATPase from the salivary gland of the tick Amblyomma americanum (L). 901 57

Vacuolar ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. An unusual member of this family, a sodium-translocating ATPase, has been found in the eubacterium Enterococcus hirae. We report here the purification of enterococcal Na+-ATPase from the plasma membrane of cells, whose ATPase content was highly amplified by expression of the cloned ntp operon that encodes this Na+-ATPase (ntpFIKECGABDHJ). The purified enzyme appears to consist of nine Ntp polypeptides, all the above except for the ntpH and ntpJ gene products. ATPase activity was strictly dependent on the presence of Na+ or Li+ ions and was inhibited by nitrate, N-ethylmaleimide, and the peptide antibiotic destruxin B. When the purified ATPase was reconstituted into liposomes prepared from Enterococcus faecalis phospholipids, ATP-driven Na+ uptake was observed; uptake was blocked by nitrate, destruxin B, and monensin, but it accelerated by carbonyl cyanide m-chlorophenylhydrazone and valinomycin. These data demonstrate that E. hirae Na+-ATPase is an electrogenic sodium pump of the vacuolar type. This is a promising system for research on the fundamental molecular structure and mechanism of vacuolar ATPase.
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PMID:Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae. 931 89

The vma41-1 mutant was identified in a genetic screen designed to identify novel genes required for vacuolar H+-ATPase activity in Saccharomyces cerevisiae. The VMA41 gene was cloned and shown to be allelic to the CYS4 gene. The CYS4 gene encodes the first enzyme in cysteine biosynthesis, and in addition to cysteine auxotrophy, cys4 mutants have much lower levels of intracellular glutathione than wild-type cells. cys4 mutants display the pH-dependent growth phenotypes characteristic of vma mutants and are unable to accumulate quinacrine in the vacuole, indicating loss of vacuolar acidification in vivo. The vacuolar proton-translocating ATPases (V-ATPase) is synthesized at normal levels and assembled at the vacuolar membrane in cys4 mutants, but its specific activity is reduced (47% of wild type) and the activity is unstable. Addition of reduced glutathione to the growth medium complements the pH-dependent growth phenotype, partially restores vacuolar acidification, and restores wild type levels of ATPase activity. The CYS4 gene was deleted in a strain in which the catalytic site cysteine residue implicated in oxidative inhibition of the yeast V-ATPase has been mutagenized (Liu, Q., Leng, X.-H., Newman, P., Vasilyeva, E., Kane, P. M., and Forgac, M. (1997) J. Biol. Chem. 272, 11750-11756). This catalytic site point mutation suppresses the effects of the cys4 mutation. The data indicate that the acidification defect of cys4 mutants arises from inactivation of the vacuolar ATPase in the less reducing cytosol resulting from loss of Cys4p activity and provide the first evidence for the modulation of V-ATPase activity by the redox state of the environment in vivo.
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PMID:Mutations in the CYS4 gene provide evidence for regulation of the yeast vacuolar H+-ATPase by oxidation and reduction in vivo. 934 71

We have used an enhancer-trap approach to begin characterizing the function of the Drosophila endocrine system during larval development. Five hundred and ten different lethal PZ element insertions were screened to identify those in which a reporter gene within the P element showed strong expression in part or all of the ring gland, the major site of production and release of developmental hormones, and which had a mutant phenotype consistent with an endocrine defect. Nine strong candidate genes were identified in this screen, and eight of these are expressed in the lateral cells of the ring gland that produce ecdysteroid molting hormone (EC). We have confirmed that the genes detected by these enhancer traps are expressed in patterns similar to those detected by the reporter gene. Two of the genes encode proteins, protein kinase A and calmodulin, that have previously been implicated in the signaling pathway leading to EC synthesis and release in other insects. A third gene product, the translational elongation factor EF-1alpha F1, could play a role in the translational regulation of EC production. The screen also identified the genes couch potato and tramtrack, previously known from their roles in peripheral nervous system development, as being expressed in the ring gland. One enhancer trap revealed expression of the gene encoding the C subunit of vacuolar ATPase (V-ATPase) in the medial cells of the ring gland, which produce the juvenile hormone that controls progression through developmental stages. This could reveal a function of V-ATPase in the response of this part of the ring gland to adenotropic neuropeptides. However, the gene identified by this enhancer trap is ubiquitously expressed, suggesting that the enhancer trap is detecting only a subset of its control elements. The results show that the enhancer trap approach can be a productive way of exploring tissue-specific genetic functions in Drosophila.
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PMID:Genes expressed in the ring gland, the major endocrine organ of Drosophila melanogaster. 958 98

The macrolide antibiotic bafilomycin A1 is a highly potent and selective inhibitor of all the vacuolar ATPases (V-ATPases). With the aim of obtaining novel analogues specific for the osteoclast subclass of vacuolar ATPase, 31 derivatives of bafilomycin A1 were synthesized and tested for their ability to inhibit differentially the V-ATPase-driven proton transport in membrane vesicles derived from chicken osteoclasts (cOc) and bovine chromaffin granules (bCG). Although none of the new analogues were more potent than the parent compound, the obtained data provided a significant amount of information about the structural requirements for the inhibitory activity of bafilomycin A1. The different effects of a few analogues on the two enzymes could also suggest the possibility of a selective modulation of the V-ATPases in different tissues.
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PMID:Synthesis and structure-activity relationships of bafilomycin A1 derivatives as inhibitors of vacuolar H+-ATPase. 959 38

Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.
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PMID:The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits. 960 53


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