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)

A preparative proteomic approach, involving liquid phase isoelectric focusing (IEF) in combination with one-dimensional electrophoresis and electroelution followed by mass spectrometry and database searches, was found to be an important tool for identifying low-abundant proteins (microgram/L) in human cerebrospinal fluid (CSF) and membrane proteins in human frontal cortex. Several neuron-related proteins, such as amyloid precursor-like protein, chromogranins A and B, glial fibrillary acid protein, beta-trace, transthyretin, ubiquitin, and cystatin C, were identified in CSF. Several types of proteins were also characterized from a detergent-solubilized human frontal cortex homogenate including membrane proteins such as synaptophysin, syntaxin and Na+/K+ ATPase. One-third of the identified proteins have not previously been identified in human CSF or human frontal cortex using proteomic techniques. The absence of these proteins in two-dimensional electrophoresis maps might be due to insufficient amounts or low solubility. The advantages of using preparative liquid phase electrophoretic separations for identifying proteins from complex biological mixtures are speed of analysis, high loadability in the IEF separation, nondiscrimination of membrane proteins or low abundance proteins, yielding sufficient amounts for characterization by mass spectrometry. The use of this strategy in proteome studies of CSF/brain tissue is expected to offer new perspectives in studies of the pathology of neurodegenerative diseases, and reveal new potential markers for brain disorders.
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PMID:Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis approach prior to mass spectrometry. 1168 Aug 89

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function.
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PMID:Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion. 1207 Jan 32

The apical- and basolateral-specific distribution of target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H(+)-ATPase in alpha- but not beta-intercalated cells, suggesting its involvement in H(+)-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.
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PMID:SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes. 1237 88

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.
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PMID:Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis. 1265 53

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.
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PMID:The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis. 1278 20

The symbiosome membrane represents a specialized plant membrane that forms both a structural and a functional interface between the legume plant and its bacterial counterpart. In this study, the symbiosome membrane protein profile from the model system Medicago truncatula and the corresponding bacterium Sinorhizobium meliloti was examined using two-dimensional electrophoresis and microcapillary high-performance liquid chromatography (HPLC) tandem mass spectrometry. The identities of 51 proteins were obtained and these proteins were categorized into functional classes to indicate biochemical roles. Symbiosome membrane proteins include an H(+)-ATPase, ENOD16, ENOD8, nodulin-25, BiP, HSP70, PDI, multifunctional aquaporin, a putative syntaxin, and other proteins of known and unknown identity and function. The majority of the proteins identified were involved with protein destination and storage. These results allow us to understand better the biochemical composition of the symbiosome membrane and thus provide a basis to hypothesize mechanisms of symbiosome membrane formation and function.
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PMID:Biochemical characterization of symbiosome membrane proteins from Medicago truncatula root nodules. 1476 Jun 46

H(+) transport in the collecting duct is regulated by exocytic insertion of H(+)-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H(+)-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H(+)-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H(+)-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1ADeltaC [amino acids (aa) 1-264] formed complexes with H(+)-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H(+)-ATPase (aa 235-264) and another that bound SNAP23 and VAMP (aa 190-234) to an equivalent degree as full-length syntaxin. However, the aa 235-264 cassette alone without the SNARE N (aa 1-160) does not bind but requires ligation to the SNARE N to bind H(+)-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H(+)-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H(+)-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H(+)-ATPase exocytosis.
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PMID:Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells. 1587 13

Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobrevin/VAMP, the calcium sensor synaptotagmin, and the ATPase NSF. Also present is caveolin-1, a component of lipid rafts. Stallions with fertility problems presented the worst quality of sperm and acrosomal membrane, and had less sperm cells stained positively for SNAREs and caveolin-1, than sperm from fertile donors (p < 0.001). Ubiquitin surface staining was also performed and it seemed to inversely correlate with stallion fertility, supporting data obtained with the negative staining technique. A male-related problem was confirmed when mares that had failed to impregnate with samples from an infertile stallion were successfully inseminated with sperm from a fertile donor. Furthermore NSF, synaptotagmin and caveolin-1 staining seemed to be useful in predicting stallion fertility, i.e. significantly more sperm cells stained positively for these proteins in samples from fertile males. Although these results need to be expanded on a larger scale, they suggest that acrosomal and surface staining of equine sperm with novel probes may constitute useful tools in predicting stallion fertility.
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PMID:SNARE proteins and caveolin-1 in stallion spermatozoa: possible implications for fertility. 1595 53

Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians caused by the trafficking defects of CF transmembrane conductance regulator (CFTR), which is a cAMP-dependent Cl- channel at the plasma membrane. The trafficking pathway of CFTR is thought to be non-conventional because CFTR maturation is inhibited by the dysfunction of syntaxin 13, which is involved in protein recycling via endosomal pathway. In this study, to clarify whether the endosomal trafficking is required for CFTR maturation, we utilized a specific vacuolar H+-ATPase inhibitor, bafilomycin A1 (BafA1), which inhibits the protein trafficking from early endosome. Our data showed that low concentration of BafA1 (50 nM) decreased the expression of mature CFTR but induced the accumulation of immature CFTR in the juxta-nuclear region containing an early endosome marker. Pulse-chase analysis showed that BafA1 inhibited the maturation of CFTR, but it slightly stabilized immature CFTR. These results indicate that BafA1-sensitive pathway is required for CFTR maturation and emphasize that endosomal trafficking pathway might be involved in the maturation of CFTR.
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PMID:Bafilomycin A1-sensitive pathway is required for the maturation of cystic fibrosis transmembrane conductance regulator. 1700 67

To understand how rabies virus (RV) infection results in neuronal dysfunction, the authors employed proteomics technology to profile host responses to RV infection. In mice infected with wild-type (wt) RV, the expression of proteins involved in ion homeostasis was altered. H(+) ATPase and Na(+)/K(+) ATPase were up-regulated whereas Ca(2+) ATPase was down-regulated, which resulted in reduction of the intracellular Na(+) and Ca(2+) concentrations. Furthermore, infection with wt RV resulted in down-regulation of soluble NSF attachment receptor proteins (SNAREs) such as alpha-synaptosome-associated protein (SNAP), tripartite motif-containing 9 (TRIM9), syntaxin, and pallidin, all of which are involved in docking and fusion of synaptic vesicles to and with presynaptic membrane. As a consequence, accumulation of synaptic vesicles was observed in the presynapses of mice infected with wt RV. These data demonstrate that infection with wt RV results in alteration of host protein expression, particularly those involved in ion homeostasis and docking and fusion of synaptic vesicles to presynaptic membrane, which may lead to neuronal dysfunction. On the other hand, attenuated RV up-regulated the expression of proteins involved in the induction of apoptosis, explaining why apoptosis is observed only in cells or animals infected with attenuated RV in previous studies.
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PMID:Proteomic profiling reveals that rabies virus infection results in differential expression of host proteins involved in ion homeostasis and synaptic physiology in the central nervous system. 1750 79


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