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)

Ca(2+)-sensitive mutants of the yeast Saccharomyces cerevisiae showing a Pet- phenotype (cls7-cls11) have lesions in a system for maintaining intracellular Ca2+ homeostasis (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1986) J. Gen. Microbiol. 132, 979-988). Genetic and biochemical studies have demonstrated that these Pet- cls mutants are related to defects in vacuolar membrane H(+)-ATPase. CLS7 and CLS8 were found to be identical with the structural genes encoding subunit c (VMA3) and subunit a (VMA1), respectively, of the enzyme. In addition, these five mutants all had vma defects; no vacuolar membrane ATPase activity was detected in the cls cells, and the cls mutants showed a loss of ability to acidify the vacuole in vivo. Measurements of the cytosolic free Ca2+ concentration [( Ca2+]i) in individual cells showed that the average [Ca2+]i in wild-type cells was 150 +/- 80 nM, whereas that in five Pet- cls cells was 900 +/- 100 nM. These data are consistent with the observation that vacuolar membrane vesicles prepared from the Pet- cls cells have lost ATP-dependent Ca2+ uptake activities. The cls defects of vacuolar membrane H(+)-ATPase resulted in pleiotropic effects on several cellular activities, including Ca2+ homeostasis, glycerol metabolism, and phospholipid metabolism. The mutants showed an inositol-dependent phenotype, possibly due to alteration in regulation of phospholipid biosynthesis; the phosphatidylserine decarboxylase activities of the mutants were 15-50% of that of the wild-type cells and were not repressed by the addition of inositol. In contrast to the majority of previously isolated pet mutants (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225), the Pet- cls mutants showed no detectable mitochondrial defects. Taking all these findings into account, we suggest that at least six genes, VMA1 (CLS8, subunit a), VMA2 (subunit b), VMA3 (CLS7, subunit c), VMA11 (CLS9), VMA12 (CLS10), and VMA13 (CLS11), are required for expression of the vacuolar membrane H(+)-ATPase activity.
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PMID:Calcium-sensitive cls mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H(+)-ATPase activity. 183 Mar 11

Our current work on a vacuolar membrane proton ATPase in the yeast Saccharomyces cerevisiae has revealed that it is a third type of H+-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F0F1-type ATP synthase and the plasma membrane E1E2-type H+-ATPase. The vacuolar membrane H+-ATPase is composed of three major subunits, subunit a (Mr = 67 kDa), b (57 kDa), and c (20 kDa). Subunit a is the catalytic site and subunit c functions as a channel for proton translocation in the enzyme complex. The function of subunit b has not yet been identified. The functional molecular masses of the H+-ATPase under two kinetic conditions have been determined to be 0.9-1.1 x 10(5) daltons for single-cycle hydrolysis of ATP and 4.1-5.3 x 10(5) daltons for multicycle hydrolysis of ATP, respectively. N,N'-Dicyclohexyl-carbodiimide2 does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-sensitive catalytic site. Cloning of structural genes for the three subunits of the H+-ATPase (VMA1, VMA2, and VMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.
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PMID:Structure and function of the yeast vacuolar membrane proton ATPase. 253 38

The Saccharomyces cerevisiae gene, VPH1 (Vacuolar pH 1), encodes a 95-kDa integral membrane subunit of the vacuolar-type H(+)-ATPase (V-ATPase) that is required for enzyme assembly; disruption of the VPH1 gene impairs vacuolar acidification (Manolson, M.F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. Chem. 267, 14294-14303). Here we show that STV1 (Similar To VPH1) encodes an integral membrane polypeptide of 102 kDa with 54% identity with the peptide sequence of Vph1p. High copy expression of STV1 partially restores vacuolar acidification in a delta vph1 mutant strain; solubilization and fractionation of membrane proteins from these vacuoles show that Stv1p co-purifies with bafilomycin A1-sensitive ATPase activity and with the 60- and 69-kDa V-ATPase subunits. Immunofluorescence microscopy of strains bearing a single copy of epitope-tagged STV1 reveals punctate staining of the cytoplasm; overexpression of epitope-tagged Stv1p reveals both punctate cytoplasmic staining and vacuolar membrane staining. Northern analysis shows that disruption of STV1 does not affect the level of transcription of VPH1 and that disruption of VPH1 does not affect the level of transcription of STV1. Strains bearing disruption of genes encoding other V-ATPase subunits (VMA1, VMA2, VMA3, and VMA4) fail to grow on media supplemented with 100 mM CaCl2 or 4 mM ZnCl2, media buffered to pH 7.5, or media with a glycerol carbon source. On the same types of media only a delta vph1 delta stv1 double disruption mutant has growth phenotypes equivalent to strains bearing a single disruption of the VMA1, VMA2, VMA3, and VMA4 genes; a delta vph1 strain has only moderate growth inhibition while a delta stv1 strain has wild type growth on the conditions listed above. We conclude that Stv1p is a functional homologue of Vph1p and suggest that Stv1p and Vph1p may be equivalent subunits for V-ATPases located on different organelles. The function of these 100-kDa homologues may be to target or regulate other common V-ATPase subunits for two distinct cellular locations.
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PMID:STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p. 751 99

The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and to possess significant sequence homology with the alpha subunit of the mitochondrial F-ATPase, which forms the major portion of the noncatalytic nucleotide binding sites and contributes several residues to the catalytic sites of this complex. Based upon the recent x-ray structure of the mitochondrial F1 ATPase (Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994) Nature 370,621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a deletion of this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutations at two residues postulated to be contributed by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete loss of ATPase activity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the requirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of proton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in the region corresponding to the P-loop of the alpha subunit (H180K, H180G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the ability of Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.
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PMID:Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p). 856 53

To investigate residues involved in the formation of the noncatalytic nucleotide binding sites of the vacuolar proton-translocating adenosine triphosphatase (V-ATPase), cysteine scanning mutagenesis of the VMA2 gene that encodes the B subunit in yeast was performed. Replacement of the single endogenous cysteine residue at position 188 gave rise to a Cys-less form of the B subunit (Vma2p) which had near wild-type levels of activity and which was used in the construction of 16 single cysteine-containing mutants. The ability of adenine nucleotides to prevent reaction of the introduced cysteine residues with the sulfhydryl reagent 3-(N-maleimidopropionyl)biocytin (biotin-maleimide) was evaluated by Western blot. Biotin-maleimide labeling of the purified V-ATPase from the wild-type and the mutants S152C, L178C, N181C, A184C, and T279C was reduced after reaction with the nucleotide analog 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (BzATP). These results suggest the proximity of these residues to the nucleotide binding site on the B subunit. In addition, we have examined the level of endogenous nucleotide bound to the wild-type V-ATPase and to a mutant (the A subunit mutant R483Q) which is postulated to be altered at the noncatalytic site and which displays a marked nonlinearity in ATP hydrolysis (MacLeod, K. J., Vasilyeva, E., Baleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156). The R483Q mutant contained 2.6 mol of ATP/mol of V-ATPase compared with the wild-type enzyme, which contained 0.8 mol of ATP/mol of V-ATPase. These results suggest that binding of additional ATP to the noncatalytic sites may modulate the catalytic activity of the enzyme.
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PMID:Cysteine scanning mutagenesis of the noncatalytic nucleotide binding site of the yeast V-ATPase. 1061 13

Several of the 13 subunits comprising mammalian H(+)-ATPases have multiple isoforms, encoded by separate genes and with differing tissue expression patterns, which may play an important role in the intracellular localization and activity of H(+)-ATPases. Here we report the cloning of three previously uncharacterized human genes, ATP6V1C2, ATP6V1G3 and ATP6V0D2, encoding novel H(+)-ATPase subunit isoforms C2, G3 and d2, respectively. We demonstrate that these novel genes are expressed in kidney and few other tissues, and confirm previous reports that the C1, G1 and d1 isoforms are ubiquitously expressed, while G2 is brain-specific. Previously we have shown that mutations in two kidney-specific genes, ATP6V1B1 and ATP6V0A4, encoding the H(+)-ATPase B1 and a4 subunit isoforms, cause recessive distal renal tubular acidosis (dRTA). As the genes reported here are expressed mainly in kidney, we assessed their candidacy as causative genes for recessive dRTA in eight kindreds unlinked to either known disease locus. Although no potential disease-causing mutations were seen in this cohort, this does not rule out a role for these genes in other families. The identification of these three novel tissue-specific isoforms supports the hypothesis that subunit differences may play a key role in the structure, site and function of H(+)-ATPases within the cell.
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PMID:Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H(+)-ATPase C, G and d subunits, and their evaluation in autosomal recessive distal renal tubular acidosis. 1238 98

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.
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PMID:Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss. 1241 17

The rare bone thickening disease osteopetrosis occurs in various forms, one of which is accompanied by renal tubular acidosis (RTA), and is known as Guibaud-Vainsel syndrome or marble brain disease. Clinical manifestations of this autosomal recessive syndrome comprise increased bone density, growth failure, intracerebral calcification, facial dysmorphism, mental retardation, and conductive hearing impairment. The most common cause is carbonic anhydrase II (CAII) deficiency. Several different loss of function mutations in CA2, the gene encoding CAII, have been described. To date, there have been no exceptions to the finding of CAII deficiency in patients with coexistent osteopetrosis and RTA. Most often, the RTA is of mixed proximal and distal type, but kindreds are reported in which either distal or proximal RTA predominates. We report the molecular genetic investigation of two consanguineous kindreds where osteopetrosis and distal RTA (dRTA) were both manifest. One kindred harbours a novel homozygous frameshift alteration in CA2. In the other, CAII levels were normal despite a similar clinical picture, and we excluded defects in CA2. In this kindred, two separate recessive disorders are penetrant, each affecting a different, tissue specific subunit of the vacuolar proton pump (H(+)-ATPase), providing a highly unusual, novel genetic explanation for the coexistence of osteopetrosis and dRTA. The osteopetrosis is the result of a homozygous deletion in TCIRG1, which encodes an osteoclast specific isoform of subunit a of the H(+)-ATPase, while the dRTA is associated with a homozygous mutation in ATP6V1B1, encoding the kidney specific B1 subunit of H(+)-ATPase. This kindred is exceptional firstly because the coinheritance of two rare recessive disorders has created a phenocopy of CAII deficiency, and secondly because these disorders affect two different subunits of the H(+)-ATPase that have opposite effects on bone density, but which have only recently been determined to possess tissue specific isoforms.
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PMID:A phenocopy of CAII deficiency: a novel genetic explanation for inherited infantile osteopetrosis with distal renal tubular acidosis. 1256 20

The multisubunit vacuolar-type proton-translocating ATPases (H(+)-ATPases) mediate the acidification of various intracellular organelles. In a subset of tissues, they also mediate H(+) secretion at the plasma membrane. Two isoforms of the H(+)-ATPase B-subunit exist in humans; we have shown that mutations in ATP6V1B1, encoding the B1-isoform, cause the clinical condition distal renal tubular acidosis. Here we report the cloning and characterization of murine Atp6v1b1, which encodes a 513-amino acid (aa) protein with 93% identity to human ATP6V1B1. Genomic organization is conserved between the murine and human H(+)-ATPase B1-subunits, and Atp6v1b1 maps to a region of mouse chromosome 6 syntenic to human 2p13, the location of ATP6V1B1. Northern blotting detects a 2.2-kb Atp6v1b1 transcript in the kidney and testis, but not other major organs. In mouse kidney, the B1-subunit localizes to intercalated cells of the cortical and medullary collecting duct. B1 protein levels were not increased in either mouse renal cortex or medulla after either 2 or 7 days of oral acid loading. These results demonstrate that Atp6v1b1 encodes the murine ortholog of human ATP6V1B1 and provides a tool for future development of animal models based on manipulation of the Atp6v1b1 genomic locus.
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PMID:Molecular cloning and characterization of Atp6v1b1, the murine vacuolar H+ -ATPase B1-subunit. 1458 95

The kidney plays a major role in maintaining and controlling systemic acid-base homeostasis by reabsorbing bicarbonate and secreting protons and acid-equivalents, respectively. During postnatal kidney development and adaptation to changing diets, plasma bicarbonate levels are increasing, the capacity for urinary acidification maturates, and the final morphology and distribution of intercalated cells is achieved. In adult kidney, at least two types of intercalated cells (IC) are found along the collecting duct characterised either by the expression of AE1 (type A IC) or pendrin (non-type A IC) where non-type A IC are found only in the convoluted distal tubule, connecting tubule and cortical collecting duct. Here we investigated in mouse kidney the relative mRNA abundance, protein expression levels and distribution of several proteins involved in renal acid-base transport, namely, the Na(+)/HCO(3)(-) cotransporter NBC1 (SLC4A4), the Na(+)/H(+)-exchanger NHE3 (SLC9A3), two subunits of the vacuolar H(+)-ATPase [ATP6V0A4 (a4), ATP6V1B1 (B1)], the Cl(-)/HCO(3)(-) exchangers AE1 (SLC4A1) and pendrin (SLC26A4). Relative mRNA abundance of all transport proteins was lowest at day 3 after birth and increased thereafter in parallel with protein levels. The numbers of type A and non-type A IC in the cortical collecting duct (CCD) increased from day 3 to days 18 and 24, whereas the number of IC in the CCD with apical staining for the vacuolar H(+)-ATPase subunits a4 and B1 decreased from day 3 to days 18 and 24, respectively. In addition, cells with characteristics of non-type A IC (pendrin expression, basolateral expression of vacuolar H(+)-ATPase subunits) were found in the inner and outer medulla 3 days after birth but were absent from the medulla of 24-day-old mice. Taken together, these results demonstrate massive changes in mRNA and protein expression levels of several acid-base transporters during postnatal kidney maturation and also show changes in intercalated cell phenotype in the medulla during these processes.
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PMID:Postnatal expression of transport proteins involved in acid-base transport in mouse kidney. 1475 80


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