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)

Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.
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PMID:Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex. 252 96

Organelle acidification plays a demonstrable role in intracellular protein processing, transport, and sorting in animal cells. We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface. We have also determined that a subset of the vpl mutants, which are deficient in sorting of vacuolar proteins (Rothman, J. H., and T. H. Stevens. 1986. Cell. 47:1041-1051; Rothman, J. H., I. Howald, and T. H. Stevens. EMBO [Eur. Mol. Biol. Organ.] J. In press), failed to accumulate the lysosomotropic fluorescent dye quinacrine within their vacuoles, mimicking the phenotype of wild-type cells treated with ammonium. The acidification defect of vpl3 and vpl6 mutants correlated with a marked deficiency in vacuolar ATPase activity, diminished levels of two immunoreactive subunits of the protontranslocating ATPase (H+-ATPase) in purified vacuolar membranes, and accumulation of the intracellular portion of PrA as the precursor species. Therefore, some of the VPL genes are required for the normal function of the yeast vacuolar H+-ATPase complex and may encode either subunits of the enzyme or components required for its assembly and targeting. Collectively, these findings implicate a critical role for acidification in vacuolar protein sorting and zymogen activation in yeast, and suggest that components of the yeast vacuolar acidification system may be identified by examining mutants defective in sorting of vacuolar proteins.
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PMID:Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins. 252 33

We have explored the structure and subunit composition of the vacuolar ATPase of Neurospora crassa by investigating the effects of nitrate. Inhibition of enzyme activity by nitrate was correlated with dissociation of a complex of peripheral polypeptides from the integral membrane part of the enzyme. Surprisingly, this nitrate-induced release of subunits occurred only when nucleotides such as ADP, ATP, or ITP were present. ATPase inhibitors that have been proposed to act at the active site prevented release of subunits. Six polypeptides, 67, 57, 51, 48, 30, and 16 kDa, were coordinately released from the vacuolar membrane. When analyzed by size exclusion chromatography or by centrifugation through glycerol gradients, the six polypeptides behaved as an aggregate of about 440,000 kDa. We also examined vacuolar membranes by electron microscopy, using negative staining. We observed a high density of "ball and stalk" structures on the membranes, similar in size but different in shape from the F0F1-ATPase of mitochondrial membranes. Treatment with nitrate removed the ball and stalk structures from vacuolar membranes but had no visible effect on mitochondrial membranes. We concluded that the overall structure of the vacuolar ATPase is similar to that of F0F1-ATPases; however, the sizes of the component polypeptides and the factors that can cause dissociation are different.
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PMID:The vacuolar ATPase of Neurospora crassa contains an F1-like structure. 252 54

Unlike most tonoplast ATPases, the vacuolar ATPase of Acer pseudoplatanus cells (Km = 0.4 mM) was strongly inhibited by vanadate (I50 = 10 microM). The inhibition was non-competitive. Chemicals usually added in the reaction mixture either increase (NH+4, K+) or decrease (Na+, EDTA) the ATPase inhibition. However, these results do not explain the insensitivity to vanadate of most tonoplast ATPases. We suggest that the tonoplast contains 2 classes of ATPases, one sensitive to vanadate, the other insensitive; each class should be more or less abundant (or active) according to the plant species studied or its physiological state of growth. It appears from this study that sensitivity or insensitivity of an ATPase to vanadate is not really a good criterion to distinguish between plasmalemma and tonoplast.
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PMID:Inhibition of the vacuolar ATPase of Acer pseudoplatanus cells by vanadate. 252 80

Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump.
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PMID:Cholinergic synaptic vesicles contain a V-type and a P-type ATPase. 255 14

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.
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PMID:Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-2 encoding the 57-kDa polypeptide and comparison to vma-1. 284 51

Polyacrylamide gel electrophoresis (PAGE) of partially purified ATPase from vacuoles of Saccharomyces carlsbergensis under non-dissociating conditions revealed 3 bands with ATPase activity. Further PAGE in dissociating conditions showed the similarity in composition of these 3 ATPase preparations. They were assumed to contain the same vacuolar ATPase exhibiting, however, different electrophoretic mobility due to the formation of enzyme complexes with different proteins and phospholipids. The ATPase preparation with the highest electrophoretic mobility contained 6 subunits of 75, 62, 16, 14, 12 and 9 kDa. Inhibitors of vacuolar ATPase [14C]DCCD and [14C]NEM bound to a 9 kDa polypeptide, while [14C]DES associated with a polypeptide of 75 kDa. A partially purified preparation of the vacuolar ATPase was not phosphorylated by [gamma-32P]ATP under conditions when plasma membrane ATPase formed a phosphorylated intermediate. Our results show that vacuolar H+-ATPase consists of several polypeptides, does not form the phosphorylated intermediate, and evidently represents a new type of H+-ATPase of yeast.
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PMID:What family of ATPases does the vacuolar H+-ATPase belong to? 286 63

Vacuolar ATPases constitute a novel class of N-ethylmaleimide- and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble F0F1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the approximately 70-kDa subunit. Using antibodies to the approximately 70-kDa subunit of corn to screen a carrot root lambda gt11 cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative F0F1-ATPase beta-chains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to beta-chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the F0F1-type ATPases. A major functional difference between the 69-kDa and beta-subunits is the location of 3 critical cysteine residues: two in the putative catalytic region (Cys-248 and Cys-256) and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the amino-terminal end of the subunit.
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PMID:The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H+-ATPase. Homology to the beta-chain of F0F1-ATPases. 289 65

Kidney cortex microsomes enriched in Golgi markers and probably also containing endosomes were isolated by cell fractionation and found to contain a proton-translocating ATPase that was inhibited by N-ethylmaleimide (NEM). This NEM-sensitive ATPase was solubilized with n-octyl glucoside and purified using anion-exchange sievorptive chromatography on sequential DEAE-Sephadex and QAE-Sephadex columns followed by a final hydroxyapatite HPLC column. The purified enzyme, with a specific activity of 4.4 mumol.mg-1.min-1 was completely inhibited by NEM. Addition of asolectin and removal of the detergent by dialysis resulted in reconstitution of NEM-sensitive electrogenic proton transport. This vacuolar ATPase is composed of five polypeptides with apparent molecular masses of 68, 58, 40, 37, and 16 kDa.
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PMID:Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes. 290 77

The vacuolar membrane of Neurospora crassa contains a H+-translocating ATPase composed of at least three subunits with approximate molecular weights of 70,000, 60,000, and 15,000. Both genomic and cDNA clones encoding the largest subunit, which appears to contain the active site of the enzyme, have been isolated and sequenced. The gene for this subunit, designated vma-1, contains six small introns (60-131 base pairs) and encodes a hydrophilic protein of 607 amino acids, Mr 67,121. Within the sequence is a putative nucleotide-binding region, consistent with the proposal that this subunit contains the site of ATP hydrolysis. This 67-kDa polypeptide shows high homology (62% identical residues overall and 84% in the middle of the protein) to the analogous polypeptide of a higher plant vacuolar ATPase. The hypothesis that the vacuolar ATPase is related to F0F1 ATPases is strongly supported by the finding of considerable homology between the 67-kDa subunit of the Neurospora vacuolar ATPase and both the alpha and beta subunits of F0F1 ATPases.
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PMID:Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases. 297 51


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