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1.
Ki-Seok Yoon Yukiko Sakai Natsuki Tsukada Kiyoshi Fujisawa & Hirofumi Nishihara 《FEMS microbiology letters》2009,290(1):114-120
Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H2 oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H2 oxidation/H2 evolution) was 1.61 × 102 at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H2 . Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V max =336 U mg−1 , k cat =560 s−1 , and k cat / K m =2.24 × 107 M−1 s−1 . The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]+ and [4Fe–4S]+ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H2 -oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga . 相似文献
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The effects of external K+ , H+ and Ca2+ concentrations on the intracellular K+ concentration, [K+ ]i, and the K+ -ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K+ ]i, in mung bean roots was markedly decreased by external K+ or H+ stress and did not recover the initial value even after the stress was removed. This decrease in [K+ ]i, gradually disappeared with the addition of (Ca2+ . Ca2+ may offset the harmful effects of ion stress. Ca2+ seems to have two effects on K+ transport; control of K+ permeability and activation of K+ uptake, although K+ -ATPase activity was inhibited by Ca2+ concentrations higher than 10–4 M. We suggest that Ca2+ activates K+ uptake indirectly through the acidification of the cytoplasm. 相似文献
4.
Q H Tran T Arras S Becker G Holighaus G Ohlberger G Unden 《European journal of biochemistry》2000,267(15):4817-4824
The oxygen sensor regulator FNR (fumarate nitrate reductase regulator) of Escherichia coli is known to be inactivated by O2 as the result of conversion of a [4Fe-4S] cluster of the protein into a [2Fe-2S] cluster. Further incubation with O2 causes loss of the [2Fe-2S] cluster and production of apoFNR. The reactions involved in cluster assembly and reductive activation of apoFNR isolated under anaerobic or aerobic conditions were studied in vivo and in vitro. In a gshA mutant of E. coli that was completely devoid of glutathione, the O2 tension for the regulatory switch for FNR-dependent gene regulation was decreased by a factor of 4-5 compared with the wild-type, suggesting a role for glutathione in FNR function. In isolated apoFNR, glutathione could be used as the reducing agent for HS- formation required for [4Fe-4S] assembly by cysteine desulfurase (NifS), and for the reduction of cysteine ligands of the FeS cluster in FNR. Air-inactivated FNR (apoFNR without FeS) could be reconstituted to [4Fe-4S].FNR by the same reaction as used for apoFNR isolated under anaerobic conditions. The in vivo effects of glutathione on FNR function and the role of glutathione in the formation of active [4Fe-4S].FNR in vitro suggest an important role for glutathione in the de novo assembly of FNR and in the reductive activation of air-oxidized FNR under anaerobic conditions. 相似文献
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Janine M. Barnes Paul A. Murphy David Kirkhara Jeremy M. Henley 《Journal of neurochemistry》1993,61(5):1685-1691
Abstract— Recent reports have suggested that a major proportion of [3 H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [3 H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [3 H]kainate binding and a 50% reduction in the apparent B max values in both Mg2+ /Na+ and Mg2+ /Na+ -free buffer when assayed at 0°c. The pharmacology of [3 H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [3 H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[3 H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [3 H]kainate and 6-[3 H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation. 相似文献
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Abstract The Escherichia coli ptsI and ptsH genes code for the synthesis of two proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), namely enzyme I and protein HPr. A number of ptsI + ptsH + /F' ptsI + ptsH merodiploids was obtained. It was shown in experiments in vivo that ptsH mutations in the transposition are dominant. Bacterial extracts from these merodiploids supported [14 C]methyl glucoside (MG) phosphorylation at the expense of phosphoenolpyruvate only half as much as extracts from the pts + cells. ptsI + ptsH /F' ptsI + ptsH + merodiploids appeared to be non-viable; the reason for this lack of viability is discussed. 相似文献
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SYNTHESIS AND RELEASE OF [14 C]ACETYLCH0LINE IN SYNAPTOSOMES 总被引:4,自引:2,他引:2
T. Haga 《Journal of neurochemistry》1971,18(6):781-798
Abstract— Synaptosomes took up [14 C]choline, about half or more of which was converted to [I4 C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[14 C] choline and neostigmine. The amount of [14 C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na+ concentration in the incubation medium. The effect of Na+ on the uptake of [I4 C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [14 C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K+ concentration in the medium from 5 m m to 35 m m . The change of Na+ concentration hardly affected the release of [14 C]acetylcholine. The effect of K+ on the release of [14 C]choline was rather small compared to that on [14 C] acetylcholine. Ouabain promoted the release of [14 C]acetylcholine. 相似文献
About 25 per cent of [
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Abstract— 45 Ca2+ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K+ at all times investigated. The accumulated 45 Ca2+ was bound within the synaptosome. 45 Ca2+ -ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal 45 Ca2+ then returned to control values. A23187 also increased intra-synaptosomal Na+ and Cl− contents. Botulinum toxin inhibits the K.+ -stimulated release of [14 C]ACh from synaptosomes but the ionophore released [14 C]ACh from both normal and botulinum-treated preparations in a Ca2+ -dependent manner. However, it also elicited Ca2+ -dependent release of [choline. Increased extracellular Ca2+ (10 mM and 20 mM) released [14 C]ACh (but not [14 C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca2+ essential for the mechanism of ACh release. 相似文献
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Tomoyoshi Uchikawa Yuji Kiuchi Akihiko Yura Noriyuki Nakachi Yukako Yamazaki Chie Yokomizo Katsuji Oguchi 《Journal of neurochemistry》1995,65(5):2065-2071
Abstract: We studied effects of Ca2+ in the incubation medium on [3 H]dopamine ([3 H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca2+ (0–30 min) and Ca2+ concentration (0–10 m M ) in Krebs-Ringer medium affected [3 H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca2+ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V max of the [3 H]DA uptake process. On the other hand, [3 H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca2+ . The Ca2+ -dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca2+ -free medium following preincubation with 1 m M Ca2+ . Protein kinase C inhibitors did not affect apparently Ca2+ -dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca2+ /calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca2+ -dependent enhancement of [3 H]DA uptake is mediated by activation of calmodulin-dependent protein kinases. 相似文献
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Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[3 H]tropane ([3 H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [3 H]CFT binding to synaptosomal P2 membranes requires low concentrations of Na+ (peak binding between 20 and 50 m M Na+ ), is stimulated by phosphate anion or l- , but is unaffected or only slightly affected by F- , Cl- , Br- , NO3 - , or SO4 2- , Concentrations of Na+ of >50 m M become inhibitory except in the presence of l- , which shifts peak binding levels toward higher Na+ concentrations and also elevates the peak binding level. K+ strongly decreased [3 H]CFT binding with a shallow inhibition curve, and Na+ could not overcome this effect. Saturation analysis of [3 H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined. 相似文献
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Abstract: The effect of Zn2+ on t -[3 H]butylbicycloorthobenzoate ([3 H]TBOB) binding to the GABAA receptor complex was studied autoradiographically in rat brain. Zn2+ inhibited [3 H]TBOB binding in a dose-dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn2+ had regional heterogeneity; regions showing the greatest inhibition of [3 H]TBOB binding were cortical laminae I–III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [3 H]TBOB binding included cortical laminae V–VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn2+ and those of other GABAA ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [3 H]TBOB binding seemed to be additive. Ni2+ , Cd2+ , and Cu2+ also inhibited [3 H]TBOB binding with a regional heterogeneity similar to that produced by Zn2+ . These results are consistent with Zn2+ acting at the previously detected recognition site on the GABAA receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn2+ effect may reflect differential regional distribution of GABAA receptor subtypes among brain regions. Other divalent cations probably act at the Zn2+ binding site. 相似文献
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J. N. Davis 《Journal of neurochemistry》1977,28(5):1043-1050
Abstract— Crude striatal synaptosomes were used to determine the affinity of brain tyrosine 3-mono oxygenase for oxygen. The rate of tyrosine hydroxylation was determined at several different oxygen concentrations using L-[14 C]tyrosine as substrate and measuring the [14 C]DOPA formed in the presence of a decarboxylase inhibitor. The accumulation of [14 C]tyrosine by synaptosomes was unaffected by incubation in nitrogen or 2.3% oxygen. Preincubation in nitrogen for up to 4 h did not impair the ability of synaptosomes to hydroxylate tyrosine when returned to air. Furthermore, pre-incubation in either nitrogen or air produced similar changes in the ultrastructural appearance of the synaptosomes and mitochondria in the crude preparation used in these studies. Thus tyrosine hydroxylation in these synaptosomes appeared to reflect tyrosine 3-mono oxygenase activity over a range of oxygen concentrations. The apparent Km for oxygen was 2 × 10−5 at pH 6.7 and 7.4. The apparent Km was not significantly altered by the addition of Ca2+ , and was slightly increased in the presence of N 6 -mono-butyryl cyclic AMP or high K+ . These data are consistent with the hypothesis that the availability of oxygen may limit catecholamine synthesis in the intact rat brain. 相似文献
15.
Elisabeth Hansson Ingemar Jacobson Richard Venema Åke Sellström 《Journal of neurochemistry》1980,34(3):569-573
Abstract: The lipophilic cation [3 H]triphenylrnethylphosphonium bromide ([3 H]TPMP+ ) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [3 H]TPMP+ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [3 H]y-aminobutyric acid ([3 H]GABA) transport. In some experiments the distribution of 86 RbZ+ and [3 H]TPMP+ was changed upon incubation in the presence of elevated levels of K+ , ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated. 相似文献
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Abstract— The present study was undertaken to characterize the cholinergic system of primary cell cultures of mouse and rat CNS.
In confirmation of previous reports, primary cultures were found to contain choline acetyltransferase (ChAc). Furthermore they contain acetylcholine (ACh) as measured by two different bioassays. They also synthesize [3 H]ACh from [3 H]Choline offered to the cultures.
The formation of [3 H]ACh is inhibited in the presence of hemicholinium-3 (10−6 m ) to 50% or ouabain (10−3 m ) to 20% of the values found in untreated cultures. Omission of Na + from the incubation solution also diminishes the [3 H]ACh formation of the cells.
[3 H]ACh is released upon depolarisation by K+ ions in a concentration dependent manner. The release can be prevented by lack of Ca2+ ions in the incubation solution. 相似文献
In confirmation of previous reports, primary cultures were found to contain choline acetyltransferase (ChAc). Furthermore they contain acetylcholine (ACh) as measured by two different bioassays. They also synthesize [
The formation of [
[
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Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+ , Mg2+ , Hg2+ , Li+ , K+ , and Na+ were assessed on the binding of [3 H]mazindol and [3 H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn2+ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3 H]mazindol; Mg2+ (0.1–100 µ M ) had no effect; Hg2+ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [3 H]mazindol than [3 H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li+ and K+ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21°C and Hg2+ at 0°C. 相似文献
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Seitaro Ohkuma Hidehiko Narihara Masashi Katsura Takeshi Hasegawa Kinya Kuriyama 《Journal of neurochemistry》1995,65(3):1109-1114
Abstract: The functional significance of peroxynitrite in the release of [3 H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [3 H]GABA release in a dose-dependent manner. These increases in [3 H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [3 H]GABA by the formation of NO. Two types of superoxide scavengers, Cu2+ /Zn2+ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [3 H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [3 H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [3 H]GABA release from the neurons. These results indicate that NO-induced [3 H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide. 相似文献
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Abstract— Calcium is transported at a fast rate of 410 mm/day in cat sciatic nerve on injection of 45 Ca2+ into the L7 dorsal root ganglia. Nerve segments corresponding to the crest and the plateau regions of transported activity were analyzed by column chromatography on Sephadex G-100 and Biogel A 5m columns and the fast transported 45 Ca2+ found to be bound to a protein of 15,000 dalton. Using [3 H]leucine as a precursor, a labeled calcium binding protein (CaBP) was found located at the same position in elution volumes from the columns as was the protein-bound 45 Ca2 + . The level of [3 H]-labeled CaBP in the crest and plateau regions were compared using column chromatography and polyacrylamide gel electrophoresis techniques and approx 3×4 times more [3 H]-labeled activity was found in the crest as compared to the plateau. These findings indicate that Ca2+ is fast transported in association with the CaBP. The relation of CaBP to the transport filament model of axoplasmic transport and its possible role in nerve are discussed. 相似文献
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Abstract: Following incubation with [14 C]y-aminobutyric acid (GABA) or [3 H]dopamine, slices of rat striatum were superfused with media containing 36 mM K+ or ethylenediamine (EDA), 1 or 5 mM. Both K+ and EDA induced a release of [14 C]GABA, the K+ -induced release being largely Ca2+ -dependent, while the EDA-induced release was not. Whereas K+ also evoked a Ca2+ -dependent release of [3 H]dopamine, EDA evoked no release of dopamine. EDA may therefore have potential as a specific GABA releasing agent. 相似文献