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)

Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.
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PMID:Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits. 792 76

The Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of at least 10 subunits belonging to either the peripheral V1 or integral membrane V0 subcomplex. We have characterized a novel 14-kDa V-ATPase subunit (Vma7p), encoded by the VMA7 gene, which exhibits features common to both V1 and V0 subunit proteins. Vma7p is a hydrophilic protein of 118 amino acids with a predicted molecular mass of 13,452 Da. Vacuolar membranes isolated from a vma7 delta null mutant contained no V-ATPase activity. Western analysis of vma7 delta cells revealed greatly reduced levels of the remaining V0 complex V-ATPase subunits, but normal levels of the V1 subunits. However, the V1 subunits failed to associate with the vacuolar membrane. Unlike the integral membrane subunits of the V0 complex, Vma7p was easily stripped from vacuolar membranes. Density gradient fractionation revealed that Vma7p associated only with the fully assembled V-ATPase and did not associate with a separate lower density V0 subcomplex fraction. The unique properties of the Vma7p may reflect a critical role in stabilizing the V0 complex and bridging the V1 and V0 complexes to form a functional V-ATPase complex.
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PMID:VMA7 encodes a novel 14-kDa subunit of the Saccharomyces cerevisiae vacuolar H(+)-ATPase complex. 792 8

The catalytic domain of the vacuolar proton ATPase is composed of a hexamer of three A subunits and three B subunits. Here we describe the cloning and characterization of a cDNA isoform of subunit B, HO57, from an osteoclastoma cDNA library. HO57 is represented by three species of mRNA of 1.6, 2.6 and 2.8 kb and is expressed at low levels in a range of human tissues, but at significantly higher levels in brain, kidney and osteoclastoma, and is probably an ubiquitously expressed isoform. In contrast, the kidney-specific isoform has an mRNA of 2 kb and is specifically expressed at high levels only in kidney and, at a lower level, in placenta. Thus the HO57 isoform is integral to the vacuolar ATPase found in the general secretory system of all cells as well as in vacuolar-ATPase-rich sources such as neurones and osteoclasts, whereas both the kidney-specific isoform and HO57 are highly expressed in the kidney. Furthermore, we show by in situ hybridization that HO57 is the only isoform that is exclusively and highly expressed by osteoclasts.
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PMID:Heterogeneity of vacuolar H(+)-ATPase: differential expression of two human subunit B isoforms. 794 39

The Gram-positive bacteria Enterococcus hirae expel sodium by two systems: a Na+/H(+)-antiporter and a vacuolar-type Na(+)-ATPase. We isolated a mutant, NalkA, defective in the Na(+)-ATPase. NalkA grew normally at neutral pH but was unable to grow in the presence of > 100 mM sodium at pH 9.5. By functional complementation at high pH, we cloned pES1, a plasmid from an E. hirae gene bank containing a 5.2-kilobase pair region of genomic DNA. The genomic DNA in pES1 contains five complete open reading frames, ntpM, -N, -O, -P, and -Q, encoding proteins of 75, 16, 23, 38, and 11 kDa. A sixth incomplete open reading frame, ntp 'L, precedes ntpM. The 3'-end of the cloned DNA overlaps with a previously published sequence encoding the ntpA and ntpB subunits of the E. hirae Na(+)-ATPase (Takase, K., Yamato, I., and Kakinuma, Y. 1993) J. Biol. Chem. 268, 11610-11616). The insert of pES1 therefore represents the upstream region of the ntp operon that encodes the E. hirae Na(+)-ATPase. Complementation analysis with various deletions derived from pES1 suggest that the original mutation is in the ntpM gene. Of the new genes described here, three exhibited significant sequence similarity to known proteins; ntpM shares 24% identical amino acid residues with the "116-kDa" subunits of eukaryotic vacuolar ATPases, ntpN exhibits 28% sequence identity with the 16-kDa proteolipid of human vacuolar ATPase, and ntpO has sequence homology to the 31-kDa subunit of the bovine kidney vacuolar ATPase. No known proteins with sequence similarity to ntp'L, -P, or -Q could be identified. Disruption of either ntpM, -N, or -O in wild-type cells by cassette mutagenesis resulted in mutants unable to effect ATP-driven sodium extrusion. NtpM, -N, and -O therefore represent three new gene products involved in sodium extrusion by the vacuolar-type Na(+)-ATPase of E. hirae, and three more gene products, NtpL, -P, and -Q, may also be constituents of this enzyme. The ntp operon thus contains at least eight genes.
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PMID:Operon of vacuolar-type Na(+)-ATPase of Enterococcus hirae. 814 30

The hydrophobic 16-kDa polypeptide which forms gap-junction-like structures in the crustacean Nephrops norvegicus is a member of a highly conserved family of proteolipids involved in a variety of membrane transport functions in eukaryotic cells. This family also includes the product of the Saccharomyces cerevisiae VMA3 gene which encodes an integral membrane component of the vacuolar membrane H(+)-ATPase. The cDNA for the Nephrops proteolipid complements a mutation in the yeast VMA3 gene, resulting in assembly of a hybrid H(+)-ATPase comprising yeast catalytic subunits and Nephrops integral membrane components. The hybrid vacuolar ATPase was capable of ATP hydrolysis which was coupled to proton translocation and showed inhibitor binding and enzymological properties similar to those of wild-type V-ATPases (Km for ATP, 0.4 mM), suggesting that both yeast and crustacean proteolipids share conserved structure at regions of protein interaction. To facilitate isolation of the Nephrops proteolipid by affinity chromatography on a Ni(2+)-binding support, six C-terminal histidine residues were added to the proteolipid. This modification did not prohibit assembly into the hybrid H(+)-ATPase, although the resultant enzyme did have a markedly elevated Km (1.8 mM). The membrane-bound Vo sector of the ATPase was isolated by the affinity-chromatography procedure and reconstituted into synthetic vesicles. This complex was found to be impermeable to small cations in the absence of catalytic ATPase subunits either in situ in the vacuolar membrane or in the reconstituted system. The functional significance of this impermeability and the structure/function relationships between proteolipids from different sources are discussed.
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PMID:Functional properties of a hybrid vacuolar H(+)-ATPase in Saccharomyces cells expressing the Nephrops 16-kDa proteolipid. 816

Daunomycin, an anti-neoplastic agent, is known to be sequestered by acidic organelles in normal and multidrug-resistant cells [Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesman, M.M., & Pastan, I. (1986) Cancer Res. 46, 5941-5946]. We studied the mechanism of accumulation of daunomycin into acidic organelles using chromaffin granule vesicles and proteoliposomes reconstituted with purified F-type H(+)-ATPase as model systems. Radiolabeled daunomycin was taken up by chromaffin vesicles upon addition of ATP. Its ATP-dependent uptake was stimulated about 1.4- to 1.8-fold by valinomycin plus K+, but was inhibited by ammonium chloride (10 mM) and nigericin plus K+. Quinidine (5 microM), verapamil (5 microM), or vanadate (0.5 mM), inhibitors of P-glycoprotein, had no effect on its uptake. Daunomycin was also taken up by liposomes reconstituted with F-type H(+)-ATPase. Furthermore, doxorubicin and vinblastine were taken up by these vesicles, whereas colchicine and rhodamine 123 were not. The accumulations of daunomycin and doxorubicin in acidic organelles of cultured cells were decreased by inhibiting vacuolar ATPase by addition of bafilomycin A1 or concanamycin A, or by increasing the internal pH by addition of nigericin. Melittin and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide dissipated the delta pH and inhibited accumulation of daunomycin in the membrane vesicles and acidic organelles in cultured cells. These results indicate that the delta pH established by vacuolar-type ATPase drives the uptake of daunomycin, doxorubicin or vinblastine into acidic organelles, and that no specific transporters are involved in their uptakes.
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PMID:ATP-dependent uptake of anti-neoplastic agents by acidic organelles. 820 70

Heparin (20-40 micrograms/ml) inhibits by 50% both the ATP hydrolase and the delta pH formation in isolated yeast vacuoles. The GTPase activity is not inhibited under these conditions. ATP prevents this inhibition, being added 10 min before heparin. Heparin suppresses the phosphorylation of vacuolar proteins in vitro. It is supposed that the protein kinase, similar to casein kinase I, participates in the regulation of activity of the vacuolar ATPase.
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PMID:[H(+)-ATPase from yeast vacuoles is inhibited by heparin]. 821 62

The precise function of subunit B of the vacuolar H(+)-ATPase class is unknown, but it is essential for proton pumping. We have previously reported the DNA sequence and predicted protein sequence of the vacuolar ATPase subunit B for Candida tropicalis (Gu, H.H., Gallagher, M.J., Rupkey, S. and Dean, G.E. (1990) Nucleic Acids Res. 18, 7446). When the Candida gene was expressed in a Saccharomyce cerevisiae delta vat2 mutant from which the homologous gene had been deleted, viability and vacuolar acidification was restored to apparently wild-type levels. The predicted identity between these two proteins is 90%. We have searched for vacuolar ATPase subunits B from other species that might show a difference in function, when expressed in yeast, relative to the endogenous gene. We have cloned an apparently full-length 1.8-kb bovine subunit B cDNA from adrenal medulla that is about 1 kb shorter than the previously reported bovine brain cDNA (Puopolo, K., Kumamoto, C., Adachi, I., Magner, R. and Forgac, M. (1992) J. Biol. Chem. 267, 3696-3706; Nelson, R.D., Guo, X.L., Masood, K., Brown, D., Kalkbrenner, M. and Gluck, S. (1992) Proc. Natl. Acad. Sci. USA 89, 3541-3545), but nearly identical throughout the coding nucleotide and protein sequences; it is only 74% identical to the Saccharomyces subunit B protein sequence. Upon expression of this cDNA in two different delta vat2 deletion strains, the bovine cDNA restored function only partially, as judged by both viability at high pH and vacuolar acidification. Current work is aimed at determining which regions of the bovine protein require alteration in order to fully restore the delta vat2 strain to wild-type acidification, with the eventual goal of identifying interactive residues between subunit B and other proteins required for pump function.
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PMID:Saccharomyces cerevisiae expression of exogenous vacuolar ATPase subunits B. 837 94

The vacuolar membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae is a multisubunit enzyme complex composed of an integral membrane V0 sector, and a peripherally associated V1 sector. Deletion of one of several structural genes for vacuolar H(+)-ATPase subunits was previously demonstrated to prevent proper assembly of the remaining V1 subunits onto the vacuolar membrane (Kane, P.M., Kuehn, M.C., Howald-Stevenson, I., and Stevens, T.H. (1992) J. Biol. Chem. 267, 447-454). A genetic screen was designed to identify new genes whose products were essential for the synthesis, assembly, and/or function of the yeast vacuolar H(+)-ATPase. Mutants were identified based on phenotypes associated with vacuolar membrane H(+)-ATPase loss of function (vma), including an inability to grow on media buffered at neutral pH. Representatives in five complementation groups were identified, including four novel mutant vma5, vma21, vma22, and vma23, all of which were defective in vacuolar ATPase enzyme activity. We report here the characterization of two genes, VMA4 and VMA5, that encode peripheral subunits of the vacuolar H(+)-ATPase. We determined that VMA5 encodes the 42-kDa subunit of the vacuolar H(+)-ATPase. The VMA4 gene, originally described by Foury (Foury, F. (1990) J. Biol. Chem. 265, 18554-18560), was determined to encode the 27-kDa subunit of the purified yeast vacuolar H(+)-ATPase. Characterization of the vma5 and vma4 mutants revealed that the 42- and 27-kDa subunits are essential for the assembly of the peripheral membrane portion of the H(+)-ATPase onto the vacuolar membrane.
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PMID:Isolation of vacuolar membrane H(+)-ATPase-deficient yeast mutants; the VMA5 and VMA4 genes are essential for assembly and activity of the vacuolar H(+)-ATPase. 841 31

vma12 mutants of the yeast Saccharomyces cerevisiae, which were originally identified as calcium-sensitive (cls) mutants that were also respiratory deficient (Pet-), have a defect in vacuolar membrane H(+)-ATPase activity (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The VMA12 gene was cloned by complementation of the growth defects of vma12 mutants. The nucleotide sequence of the gene predicts a polypeptide of 215 amino acids (25.2 kDa) with two putative membrane-spanning domains. A null vma12 mutant, constructed by chromosomal deletion of the gene, is viable but has completely lost the vacuolar membrane H(+)-ATPase activity and exhibits the same growth defects as observed for the original vma12 mutants. Synthesis and targeting of the subunits of the H(+)-ATPase in the delta vma12 mutant cells were examined by Western blotting analyses of whole cell and vacuolar membrane protein extracts. None of the peripheral membrane subunits that we analyzed (the 69-, 60-, 42-, and 27-kDa subunits) was detected in the vacuolar membrane fractions, although the cellular levels of these polypeptides appeared to be normal. The 100- and 17-kDa integral membrane subunits of the enzyme were absent or present at a substantially reduced level in mutant vacuolar membrane fractions. Anti-Vma12p antibodies recognized a vacuolar protein with the expected molecular mass of 25 kDa. However, the Vma12 protein was not detected in the vacuolar membrane ATPase complex that had been solubilized with a zwitterionic detergent, ZW3-14, and purified by glycerol gradient centrifugation (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). These results indicate that the VMA12 gene product is not a component of the active vacuolar ATPase complex and instead suggest that this protein is required during the process of assembly and/or targeting of the enzyme complex to the vacuolar membrane.
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PMID:VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae. 841 76


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