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)

Monoclonal antibodies to the axonal transport ATPase kinesin were used in an immunofluorescent study on mammalian nerves. Following crushing of the sciatic nerve and the ventral roots of adult rats, immunoreactive material was found to accumulate rapidly, mainly proximal to a crush but also, to some degree, distal to a crush. The strongest immunofluorescence was observed after incubation with the H2 antibody against the heavy subunit of kinesin. Using the cytofluorimetric scanning (CFS) procedure, the accumulated amounts were quantified and it was found that the retrogradely accumulating kinesin-like immunoreactivity (IR) was about 4-12% of the anterogradely transported kinesin-IR. The results were compared to the vesicle marker p38 (synaptophysin), which was found to accumulate to a significant extent on both sides of the crush. Cytofluorimetric scanning measurements indicated that nearly 50% of the anterogradely accumulated p38-IR was recycling to the cell body. The results demonstrate that kinesin in the living axon is affiliated with anterogradely transported organelles. Retrogradely transported organelles appeared to carry very little kinesin-IR, suggesting that kinesin may be subject to turnover, distinct from that of p38, in the distal regions of the axon.
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PMID:The axonal transport motor 'kinesin' is bound to anterogradely transported organelles: quantitative cytofluorimetric studies of fast axonal transport in the rat. 171 8

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.
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PMID:Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes. 216 11

When cytoplasmic extracts of guinea-pig myenteric neurones are submitted to centrifugal density gradient fractionation in a zonal rotor acetylcholine is bimodally distributed in the gradient, in a peak (I) rich in synaptic vesicles of the classic type and in a denser peak (II/VI) rich in densecored vesicles and vasoactive intestinal polypeptide (VIP). The putative stable synaptic vesicle markers synaptophysin (p38), vesicular proteoglycan, and Mg2+-activated ATPase were also bimodally distributed, with a peak coincident with peak I and another, broader peak embracing peak II/VI, and neighbouring peaks of other neuropeptides resolved from peak II/VI by the density gradient separation procedure used. To establish whether the stable markers, acetylcholine and VIP in peak II/VI were present in one particle or several, attempts were made to separate them by particle-exclusion chromatography and differential osmotic fragility. These were unsuccessful, leading us to conclude that the storage particles in peak II/VI contain both neurotransmitters and all three putative stable synaptic vesicle markers. It is suggested that such particles are the counterparts, in cholinergic neurones of the myenteric plexus, of the dense-cored, enkephalin- and noradrenaline-containing vesicles of certain adrenergic neurones and, like the latter, may exist in a precursor-product relationship with the classic synaptic vesicles containing the small-molecular-mass transmitters and found in the same nerve terminals.
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PMID:Characterization, by size, density, osmotic fragility, and immunoaffinity, of acetylcholine- and vasoactive intestinal polypeptide-containing storage particles from myenteric neurones of the guinea-pig. 246 36

Previous immunocytochemical work showed that the cholinergic electromotor neurones of Torpedo marmorata contain a vasoactive intestinal polypeptide-like immunoreactivity (VIPLI) that is conveyed to the terminals by axonal transport from the cell bodies where it is presumably synthesized. In extension of this work, we have now succeeded in isolating the VIPLI storage granules from both the terminals and the axons of these neurones and characterizing them morphologically and biochemically. They were readily separated from synaptic vesicles but contained several components in common that had previously been regarded as specific for synaptic vesicles. Among these were a heparan sulphate type of proteoglycan, synaptophysin, and a Mg2+-dependent ATPase. The VIPLI concentration in lobe tissue and the amount of tissue available were both insufficient to permit the isolation of granules from the electromotor cell bodies by the same technique but it was possible to establish the presence of such granules by particle-exclusion chromatography, using the stable markers mentioned above. In contrast to the VIPLI-containing granules, axonal synaptic vesicles differed from their terminal counterparts in having a very low acetylcholine content relative to stable vesicle markers: they presumably fill up on reaching the terminal where they are exposed to higher concentrations of cytoplasmic acetylcholine.
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PMID:Isolation and characterization of secretory granules storing a vasoactive intestinal polypeptide-like peptide in Torpedo cholinergic electromotor neurones. 272 32

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.
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PMID:Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure. 290 47

The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport.
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PMID:Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms. 753 59

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing synaptophysin. By monitoring cross-reactivity with anti-synaptophysin antibody, the MVs were highly purified from bovine pineal glands. The purified MVs were morphologically similar to but distinct from neuronal synaptic vesicles by their lack of synapsin I. Immunological study indicated that the MVs contained vacuolar H(+)-ATPase, synaptotagmin and synaptobrevin 2 (VAMP2). The MVs accumulated L-glutamate at the expense of ATP hydrolysis by vacuolar H(+)-ATPase. No uptakes of melatonin, serotonin, noradrenaline, gamma-aminobutyrate or acetylcholine were observed. These results indicated that the MVs are organelles for storage of L-glutamate in pinealocytes and suggested a possibility that pinealocytes transmit glutamate signals by MVs-mediated exocytosis.
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PMID:Microvesicles isolated from bovine pineal gland specifically accumulate L-glutamate. 760 13

Histochemical study indicated that the posterior pituitary possesses numerous microvesicles (MVs) containing synaptophysin, a marker protein specific for brain synaptic vesicles (Navone, F., Di Gioia, G., Jahn, R., Browning, M., Greengard, P., and De Camilli, P. (1989) J. Cell Biol. 109, 3425-2433). By monitoring cross-reactivity with anti-synaptophysin antibody, the MVs were highly purified from bovine posterior pituitaries by a combination of differential and sucrose density gradient centrifugations. The purified MVs had an average diameter of about 60 nm and were associated with synaptophysin as revealed by immunoelectron microscopy. The vesicles contained ATPase activity partially sensitive to bafilomycin A1 and to vanadate. The membrane fraction immunoisolated with anti-synaptophysin antibody also exhibited similar ATPase activity. The two ATPases could be purified separately; the vandate-sensitive enzyme was identified as a 115-kDa polypeptide immunochemically similar to chromaffin granule P-ATPase (forming phosphoenzyme intermediate), and the bafilomycin A1-sensitive ATPase showed essentially the same properties as those of vacuolar type H(+)-ATPases. Upon addition of ATP, the MVs formed an electrochemical gradient of protons and took up norepinephrine in a reserpine-sensitive manner, indicating the presence of secondary monoamine transporter coupled with vacuolar type H(+)-ATPase. No uptake of L-glutamate, gamma-aminobutyrate, glycine, or acetylcholine was observed. The identification of MVs as organelles responsible for storage of monoamines is important for understanding the physiological function of the posterior pituitary.
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PMID:Microvesicles isolated from bovine posterior pituitary accumulate norepinephrine. 774 79

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

Anti-synaptobrevin 2 immunoprecipitates obtained from freshly prepared Triton X-100 extracts of rat synaptosomes contained, in addition to synaptophysin, a 10-kDa band, which we identified by peptide sequencing and Western blotting as the c subunit of the vacuolar proton pump (V-ATPase) also called ductin or mediatophore. Ac39 and Ac116, two other transmembrane subunits of the V0 sector of the V-ATPase, were also found by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-synaptobrevin 2 immunoprecipitates obtained from frozen-thawed Triton X-100 extracts, which were greatly enriched, instead, in SNAP-25 and syntaxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophysin immunoprecipitates. Thus, the two complexes appear to be mutually exclusive. Subcellular fractionation of rat brain demonstrated that V-ATPase subunit c is localized with synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprecipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin complex suggests that this interaction may play a role in recruiting the proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which dissociates the complex and mimics a change which occurs in vivo as a prerequisite to SNARE complex formation.
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PMID:The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive, complex. 856 78


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