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)

We have discovered a ring-shaped particle of 12.5 nm diameter, 14.5S and apparent molecular weight of approximately 570,000 that displays 6-fold radial symmetry and is composed of a single kind of an acidic (pI approximately 5.5) polypeptide of Mr 97,000 (p97). Using antibodies to this protein we have detected its occurrence in a wide range of cells and tissues of diverse species from frog to man, including highly specialized cells such as mammalian erythrocytes and spermatozoa. In Xenopus laevis oocytes, the particle is found in both isolated nuclei and in manually enucleated ooplasms, which corresponds to immunofluorescence staining dispersed over both nucleoplasm and cytoplasm. The particle has a N-ethylmaleimide (NEM)-inhibitable Mg2(+)-ATPase activity, and its amino acid sequence, as deduced from cDNA clones, displays considerable homology to the mammalian NEM-sensitive fusion protein (NSF) and yeast Sec18p believed to be essential for vesicle fusion in secretory processes, indicating that these three proteins belong to the same multigene family.
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PMID:An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF. 214 Jul 70

The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required for the structural maintenance and post-mitotic regeneration of the endoplasmic reticulum. This fusion is sensitive to the thiol-alkylating agent N-ethylmaleimide. In many intracellular fusion events N-ethylmaleimide-sensitivity is associated with a homotrimeric ATPase called N-ethylmaleimide-sensitive fusion protein or NSF. The addition of cytosol containing NSF is known to restore fusion activity to N-ethylmaleimide-treated membranes. We found that the inhibition of fusion of rat liver endoplasmic reticulum membranes (microsomes) by N-ethylmaleimide was not reversed by the addition of untreated cytosol. Fusion was also unaffected by treatment with a buffer known to remove NSF from membranes. Accordingly, no membrane-associated NSF was detected by immunoblot analysis. These data suggest that microsome fusion requires an N-ethylmaleimide-sensitive component distinct from NSF. This component was tightly associated with the membranes, so we used a number of chemical probes to characterize it in situ. Its thiol groups did not appear to be part of a GTP-binding site. They showed relatively low reactivity with sodium periodate, which induces the formation of disulphide bonds between proximate thiol groups. The thiols were not protected against N-ethylmaleimide by Zn2+, a potent inhibitor of fusion which is known to efficiently co-ordinate thiol groups. To characterize the topology of the fusion-related thiol groups we used bulky thiol-specific reagents prepared by conjugating BSA or 10 kDa aminodextran to the bifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibition of fusion by these reagents indicated that these thiols are highly exposed on the membranes. This exposure might be important for the function of these groups during GTP-triggered fusion.
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PMID:Characterization of N-ethylmaleimide-sensitive thiol groups required for the GTP-dependent fusion of endoplasmic reticulum membranes. 749 17

The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring-shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity-purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.
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PMID:Isolation and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver. 780 66

N-ethylmaleimide-sensitive fusion protein (NSF), a protein necessary for vesicular docking and/or fusion, was detected immunohistochemically in pinealocytes. NSF was distributed similarly to synaptophysin and vacuolar-type H(+)-ATPase (V-ATPase), marker proteins for synaptic-like microvesicles (MVs) abundantly present in pinealocytes. A subcellular fractionation study indicated that .> 95% of NSF was present as a membrane-bound form and that some NSF was associated with MVs. Like neuronal NSF, the protein was not solubilized from membranes with either 2 mM Mg-ATP or 2% sodium carbonate, suggesting that NSF was tightly bound to the membranes. NSF was also detected in purified MVs from bovine posterior pituitaries. Since MVs are the organelles in which transmitters are stored, these results suggest that NSF is involved in the MV-mediated exocytosis of transmitters from endocrine cells.
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PMID:Localization of N-ethylmaleimide-sensitive fusion protein in pinealocytes. 854 75

Microvesicles (MVs) in endocrine cells are morphologically similar to neuronal synaptic vesicles. MVs were shown to contain proteins involved in neurotransmitter storage such as vacuolar H(+)-ATPase and neurotransmitter transporters, and ones in vesicular trafficking such as synaptobrevins and N-ethylmaleimide-sensitive fusion protein. Isolated MVs accumulate cell-specific neurotransmitters in an energy-dependent manner. Upon stimulation, the MVs may fuse with the plasma membrane and secrete the internal neurotransmitters. Thus, endocrine cells possess an MV-mediated secretion system as an intercellular signal transducing system.
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PMID:Role of endocrine cell microvesicles in intercellular chemical transduction. 872 17

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.
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PMID:Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation. 874 44

Soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAP) proteins function in Ca(2+)-regulated exocytosis. Recent work (Schiavo et al. (1996) Nature 378, 733-736) based on in vitro protein interactions has raised the possibility that alpha- and beta-SNAPs have distinct roles in exocytosis. We have examined this possibility by comparing the activities of recombinant alpha- and beta-SNAPs. Both of these proteins were able to similarly bind NSF and activate its ATPase activity but to a lesser extent than gamma-SNAP. When introduced into digitoninpermeabilised chromaffin cells, both alpha- and beta-SNAP stimulated Ca(2+)-regulated exocytosis in a MgATP-dependent manner. The dose-response relationships for these proteins were essentially the same and addition of both proteins did not lead to any further increase in exocytosis above that due to each protein alone. We conclude that alpha- and beta-SNAPs are interchangeable isoforms with similar functions in regulated exocytosis.
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PMID:Similar effects of alpha- and beta-SNAP on Ca(2+)-regulated exocytosis. 881 86

Cysteine string protein (CSP) is a 34 kDa secretory vesicle protein bearing a "J-domain" as well as a palmitoylated cysteine-rich "string" region used for membrane attachment. Mutation of the CSP gene causes impaired presynaptic neuromuscular transmission in Drosophila melanogaster, implicating CSP as part of the exocytotic protein machinery. The J-domain of CSP shares homology with the universally conserved DnaJ family, a group of proteins that act as co-chaperones with Hsc70 and its homologs. Hsc70 is an abundant neural protein with coupled protein binding and ATPase activities. We have investigated the CSP modulation of Hsc70 ATPase activity. Here we demonstrated that CSP enhances Hsc70 ATPase activity in a dose-dependent manner. CSP activation of Hsc70 was maximal ( approximately 12 times) at 1:1 stoichiometry and above. We show that a J-domain-containing fragment (amino acids 1-82) of CSP is sufficient for the activation of Hsc70. Neither CSP nor the amino-terminal fragment stimulate the activity of the isolated Hsc70 ATPase domain (amino acids 1-386). CSP does not significantly increase the activity of N-ethylmaleimide-sensitive fusion protein, another ATPase required for transport vesicle function. Our results suggest that CSP, a DnaJ family member associated with the secretory vesicle cycle regulates Hsc70 functions. Hsc70 may function within the biochemical pathways of exo- and endocytosis to promote the formation or dissociation of multimeric complexes or to regulate conformational changes.
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PMID:The cysteine string secretory vesicle protein activates Hsc70 ATPase. 882 36

alpha-SNAP [soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein] is required for fusion of transport vesicles with their target membrane. In this study, we have examined the membrane-binding properties of alpha-SNAP. We have found that in several tissues a much larger amount of alpha-SNAP per unit weight of protein is bound to membranes than is free in the cytosol. Biochemical analysis shows that a fraction of alpha-SNAP behaves in ways characteristic of hydrophobic, lipid-associated proteins. These findings suggest that membrane binding may be accounted for, at least in part, by interaction with membrane lipid. Consistent with this idea, binding of newly synthesized alpha-SNAP to brain membranes was found to be independent of functional SNAP receptors and could be accounted for by direct binding of alpha-SNAP to membrane lipid. Furthermore, membrane lipid enhanced the ability of alpha-SNAP to stimulate NSF-dependent ATPase activity.
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PMID:Evidence for interaction of the fusion protein alpha-SNAP with membrane lipid. 923 Jan 35

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.
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PMID:A novel assay reveals a role for soluble N-ethylmaleimide-sensitive fusion attachment protein in mannose 6-phosphate receptor transport from endosomes to the trans Golgi network. 934 16


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