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S Izawa

Publications and source records attributed to S Izawa.

At least 37 records · Page 2Linked to original sources
PubMed1996

Oxidative stress response in yeast: purification and characterization of glutathione reductase from Hansenula mrakii.

Glutathione reductase was purified from a yeast. Hansenula mrakii IFO 0895, to approximately 3500-fold with 59% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis, and 123 kDa by gel filtration using a calibrated Sephadex G-150 column. The Km values for glutathione disulfide and NADPH were 21.3 microM and 14.3 microM, respectively. The enzyme was most active at pH 7.5, 55 degrees C. The enzyme was stable up to 40 degrees C, and between pHs 4 and 10. The enzyme was inhibited by p-chloromercuribenzoate and metal ions such as Fe3+, Cd2+, Cu2+, and Zn2+.

Chloromercuribenzoates↗
PubMed1996

Correlation of the OSR/ZRCI gene product and the intracellular glutathione levels in Saccharomyces cerevisiae.

The OSR (oxidative-stress-resistance) gene was cloned as the gene which enhances resistance against oxidative stress caused by lipid hydroperoxide in Saccharomyces cerevisiae. The nucleotide sequence of the gene was determined and found to be identical with the zinc-resistance-conferring (ZRCI) gene, which was cloned as the gene enhancing the resistance against zinc and cadmium. A knockout mutant of the OSR gene showed increased sensitivity to lipid hydroperoxide. The intracellular glutathione content in the knockout mutant decreased approx. 40% compared with that of wild-type cells, whereas it increased 3-fold in yeast overexpressing the OSR gene. Therefore the OSR/ZRCI gene product seems to regulate the intracellular glutathione content.

Amino Acid Sequence↗
PubMed1995

Oxidative stress response in yeast: glutathione peroxidase of Hansenula mrakii is bound to the membrane of both mitochondria and cytoplasm.

The yeast Hansenula mrakii IFO 0895 induces glutathione peroxidase (GPx) when the cells are exposed to the oxidative stress such as lipid hydroperoxide, superoxide- and hydroxy radical-generating conditions. To clarify the localization of GPx in H. mrakii cell, distribution of the enzyme was investigated. After centrifugation of the yeast protoplast homogenates at 2500 x g for 10 min, 67% of total GPx activity was recovered from the supernatant (Sup. 1) and 33% was from the pellet (Pellet 1). When the Sup. 1 was fractionated by sucrose density gradient ultracentrifugation, GPx activity was essentially recovered from the mitochondria fraction. Submitochondrial localization of the enzyme showed that 95% and 2.5% of the enzyme was recovered from the inner and outer membrane, respectively. No GPx activity was detected neither in intermembrane space nor in matrix of mitochondria. On the other hand, at least 12% of total GPx activity was recovered from the purified plasma membrane which was obtained from the Pellet 1 by successive sucrose density gradient centrifugation. Thus, the GPx of H. mrakii is present in the inner and outer membrane of mitochondria as well as the plasma membrane.

Cell Membrane↗
PubMed1995

Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae.

Role of intracellular glutathione in the response of Saccharomyces cerevisiae to H2O2 was investigated. Depletion of cellular glutathione or inhibition of gamma-glutamylcysteine synthetase (GSH-I) enhanced the sensitivity to H2O2 and suppressed the adaptation to H2O2. A mutant deficient in GSH-I also showed the hypersensitivity and could not adapt to H2O2. Incubation of the cell with amino acids constituting glutathione (L-Glu, L-Cys, Gly) increased the intracellular glutathione content, and subsequently the cell acquired resistance against H2O2. These results strongly suggest that intracellular glutathione plays an important role in the adaptive response in S. cerevisiae to oxidative damage.

Adaptation, Physiological↗
PubMed1995

Effects of alcohols on the hydrolysis of colominic acid catalyzed by Streptococcus neuraminidase.

Regulation of the activity of neuraminidase of Streptococcus sp. (group K) was evaluated by examining the effects of alcohols on the hydrolysis of colominic acids catalyzed by the neuraminidase. Two kinds of alcohol binding site, activation and inhibition sites, were proposed to exist. Competitive inhibition was observed with alcohols smaller than polyethylene glycol #300 (average molecular weight: 300), so the inhibition site is considered to be the substrate binding site, the size of which was estimated to be 10 A in diameter. On the contrary, polyethylene glycols larger than this size activated the enzyme by 1.5-1.8 times. The activity could be raised by binding of the polyethylene glycols to the activation site. This activation was shown to be due solely to the decrease in the Michaelis constant, Km. The smaller polyethylene glycols (#200 and #300) were also considered to bind to the activation site, although activation was not clearly observed due to compensation with inhibition. Strong substrate inhibition by colominic acid was also observed. The activity of Streptococcus neuraminidase was shown to be regulated intricately by the substrate colominic acid and alcohols contained in the reaction medium.

Alcohols↗
PubMed1995

Characteristics of the chemical forms of 11C, 13N, and 15O induced in air by the operation of a 100 MeV electron linear accelerator.

To characterize airborne radioactivity induced by the operation of high-energy accelerators, the fractions of aerosol and gaseous components, and the chemical forms of 11C, 13N, and 15O produced in the air of a target room of a 100 MeV electron linear accelerator were studied. Measurements of radioactivity using a particulate air sampling filter and a gas flow-through ionization chamber showed that more than 98% of 11C, 13N, and 15O were present as gaseous forms. Their chemical forms, detected by means of radio-gas chromatography, were 11C as CO2; 13N as N2 and NO; and 15O as O2 and NO. Machine operating conditions, which affect the compositions of the induced radionuclides and of their chemical forms, and the resulting effect on the estimation of internal doses are discussed.

Aerosols↗
PubMed1995

Molecular cloning and nucleotide sequence of purine nucleoside phosphorylase and uridine phosphorylase genes from Klebsiella sp.

Klebsiella sp. LF 1202 was isolated as a bacterium that can assimilate adenosine as a sole source of carbon and nitrogen [F. Ling et al., Agric. Biol. Chem., 55, 573-575 (1991)] from a soil sample. Both the purine nucleoside phosphorylase (PNPase) and uridine phosphorylase (UPase) of this bacterium were induced simultaneously when the bacterium was cultured in a medium containing adenosine or uridine as a sole source of carbon and nitrogen. This induction profile is different from that of Escherichia coli. Here we cloned and sequenced the gene corresponding to each enzyme. The open reading frame (ORF) of the PNPase gene consisted of 717 bp that encoded a polypeptide of 239 amino acids with a molecular weight of 26,198. The ORF of the UPase gene consisted of 834 bp that encoded a polypeptide of 278 amino acids with a molecular weight of 28,912.

Amino Acid Sequence↗
PubMed1994

F0F1-ATPase of Vibrio parahaemolyticus: purification using new detergents and characterization.

Previous attempts to isolate a stable F0F1-ATPase complex (H(+)-translocating ATPase) from Vibrio parahaemolyticus have been unsuccessful. Using new non-ionic detergents (alkyl thiomaltosides), a stable F0F1 complex with a high specific activity (15-25 units/mg protein) was purified and characterized. The purified F0F1-ATPase consists of eight subunits (alpha, beta, gamma, delta, epsilon, a, b and c). The new detergents, in combination with sucrose (or glycerol), lipid, dithiothreitol and phenylmethylsulfonyl fluoride, effectively stabilized the F0F1 complex. The ATPase activity of the F0F1 complex was greatly increased by anions, such as SO4(2-) and SO3(2-). Sodium ion increased the activity by about 2-fold. Dicyclohexylcarbodiimide, Zn2+, 4-acetamido-4'-isothiocyanostilben-2,2'disulfonate and tetrachlorosalicylanilide inhibited F0F1-ATPase activity. Ethanol, which stimulated F1-ATPase activity, inhibited F0F1-ATPase activity. Methanol, Na3VO4 and bafilomycin A1 did not have any significant effect on F0F1-ATPase activity, although methanol, like ethanol, stimulated F1-ATPase activity.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1993

Introduction of a series of alkyl thiomaltosides, useful new non-ionic detergents, to membrane biochemistry.

We synthesized a series of non-ionic detergents, alkyl thiomaltosides, and investigated their properties and usefulness. We solubilized membrane proteins of Vibrio parahaemolyticus using the detergents. With octyl thiomaltoside, nonyl thiomaltoside, decyl thiomaltoside, or undecyl thiomaltoside, we observed satisfactory solubilization of the membrane proteins. Alkyl thiomaltosides possessing longer alkyl chains showed better solubilization than ones possessing shorter chains. H(+)-translocating ATPase (F0F1), which is localized in the cytoplasmic membrane (inner membrane), was solubilized with the detergents, and the solubilized enzyme showed much higher specific activity than that solubilized with octyl glucoside or heptyl thioglucoside, other useful non-ionic detergents. 5'-Nucleotidase, which seems to be an outer membrane protein, was also efficiently solubilized with the alkyl thiomaltosides. Membrane proteins of Escherichia coli were also efficiently solubilized with the detergents. Octyl thiomaltoside and nonyl thiomaltoside were removed fairly rapidly on dialysis. Decyl thiomaltoside was removed slowly, and undecyl thiomaltoside and dodecyl thiomaltoside were difficult to remove by dialysis.

5'-Nucleotidase↗
PubMed1992

Structural and functional modulation of the manganese cluster in Ca(2+)-depleted photosystem II induced by binding of the 24-kilodalton extrinsic protein.

Depletion of functional Ca2+ from photosystem (PS) II membranes impairs O2 evolution. Redox properties of the Mn cluster as probed by thermoluminescence were modified differently in Ca(2+)-depleted PSII depending on the procedure for Ca2+ extraction. Ca2+ depletion by low-pH treatment gave rise to an abnormally modified S2 state exhibiting a thermoluminescence band with elevated peak temperature accompanied by a marked upshift in threshold temperature for its formation, whereas Ca2+ depletion by NaCl washing in the light followed by the addition of EDTA could generate a similarly modified S2 state only when the Ca(2+)-depleted PSII was reconstituted with the 24-kDa extrinsic proteins. These results indicated that manifestation of the abnormal properties of the Ca(2+)-depleted S2 state is significantly contributed by the association of the 24-kDa extrinsic protein to PSII. It was inferred that the 24-kDa extrinsic protein regulates the structure and function of the Mn cluster in the absence of functional Ca2+ through a conformational modulation of the intrinsic protein(s) that bind(s) both Mn and Ca. Features of the extrinsic protein-dependent modulation of the Mn cluster were discussed in relation to the function of Ca2+ in O2 evolution.

Calcium↗
PubMed1983

Proton efflux coupled to dark H2 oxidation in whole cells of a marine sulfur photosynthetic bacterium (Chromatium sp. strain Miami PBS1071).

Whole cells of photoanaerobically grown Chromatium sp. strain Miami PBS1071, a marine sulfur purple bacterium, oxidized H2 in the dark through the oxyhydrogen reaction at rates of up to 59 nmol of H2 per mg (dry weight) per min. H2 oxidation was routinely measured in H2 pulse experiments with air-equilibrated cells. The reaction was accompanied by a reversible H+ efflux from the cells, suggesting an outward H+ translocation reaction coupled to H2 oxidation. The H+/e- ratio, calculated from simultaneous measurements of H2, O2, and H+ changes in the medium, varied with the cultures from 0.7 to 1.2. The ratio increased considerably when the backflow of H+ was taken into account. Anaerobic H2 uptake with 2,5-dimethyl-p-benzoguinone as an oxidant also showed a weak H+-translocating activity. No H+-translocating activity was detected with methylene blue as an oxidant. Carbonylcyanide 3-chlorophenylhydrazone (1 microM) stimulated H2 oxidation and abolished the associated H+ changes when H2 oxidation was observed in O2 pulse experiments with H2-Ar-equilibrated cells. However, the uncoupler inhibited both H2 oxidation and H+ changes when measurements were made in H2 pulse experiments with air-equilibrated cells. It is suggested that in this bacterium the susceptibility of hydrogenase to reversible O2 inactivation in situ is enhanced by the presence of uncoupling agents.

Benzoquinones↗
PubMed1980

Photophosphorylation in isolated maize bundle sheath chloroplasts and cells.

Isolated maize bundle sheath chloroplasts showed substantial rates of noncyclic photophosphorylation. A typical rate of phosphorylation coupled to whole-chain electron transport (methylviologen or ferricyanide as acceptor) was 60 mumol per hour per milligram chlorophyll) with a coupling efficiency (P/e(2)) of 0.6. Partial electron transport reactions driven by photosystem I or II supported phosphorylation with P/e(2) values of 0.2 to 0.3. Thus, two sites of phosphorylation seem to be associated with the photosynthetic chain in much the same way as in spinach chloroplasts.Isolated bundle sheath cells were capable of photosynthetic electron transport with membrane permeant electron carriers (but not with ferricyanide) at rates which were similar to those found in isolated chloroplasts. ATP formation also occurred during electron transport when ADP and phosphate were present in the cell suspension. The rates of photophosphorylation reactions in cells were about 30 to 40% of those found in isolated chloroplasts (maximum rate in cells 80 mumoles ATP per hour per milligram chlorophyll with diaminodurene as electron carrier), with the exception of endogenous photophosphorylation (photophosphorylation without added electron carriers) whose rate was three to four times higher in cells than in chloroplasts. The endogenous photophosphorylation in cells appeared to be coupled to pseudo-cyclic, rather than cyclic, electron transport. It was accompanied by O(2) uptake (when the catalase inhibitor KCN was present), was sensitive to dichlorophenyldimethylurea and methylamine, but was totally insensitive to 20 micromolar antimycin which completely inhibited succinate-supported oxidative phosphorylation in the cells. The implications of these and other phenomena associated with photophosphorylation in bundle sheath cells are discussed.

Journal Article↗
PubMed1979

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).

Chloroplasts↗
PubMed1979

Photosynthetic electron transport in isolated maize bundle sheath cells.

Fragments of bundle sheath strands, free of mesophyll cells and showing a chlorophyll a/b ratio of 6.0 to 6.6 were prepared from Zea mays by a mechanical method. They were unable to photoreduce ferricyanide but were able to photoreduce the membrane-permeant 2,5-dimethylquinone at a rate of 250 to 420 microequivalents per hour per mg chlorophyll (mueq/hr . mg Chl) at 21 C. In the presence of the catalase inhibitor KCN, methylviologen catalyzed a Mehler reaction at a rate of 120 to 180 mueq/hr . mg Chl. This was increased to 200 to 350 mueq/hr . mg Chl when the uncoupler methylamine was added. The rate of endogenous pseudocyclic electron flow, detected as a Mehler reaction, was also considerable (100 to 150 mueq/hr . mg Chl with methylamine). Diaminodurene supported a high rate of photosystem I-mediated electron flow to methylviologen (400 to 750 mueq/hr . mg Chl).When the tissue fragments were illuminated in a weakly buffered suspension, a reversible rise in the medium pH was observed which apparently originated from H(+) translocation in the thylakoids. The kinetics of the pH changes was rather slow (t((1/2)) > 15 seconds for pH rise; > 30 seconds for dark decay) but the extent of H(+) uptake was substantial (0.1 to 0.3 mueq/mg Chl). All of the electron transport reactions tested, including partial reactions which involve only photosystem I or photosystem II, invariably supported H(+) uptake. This suggests that two sites of energy conservation are associated with the photosynthetic chain in the bundle sheath chloroplasts (as in spinach chloroplasts) and that both of these sites are functional in vivo. The pH changes observed in the absence of exogenous electron carriers were abolished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea or by anaerobiosis, indicating that the underlying endogenous electron transport was strictly a pseudocyclic reaction. There was no evidence of endogenous cyclic electron flow which might contribute to the energy metabolism of the bundle sheath cells.

Journal Article↗
PubMed1978

The role of chloride ion in photosystem II. I. Effects of chloride ion on photosystem II electron transport and on hydroxylamine inhibition.

1. Chloroplasts washed with Cl--free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl- is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems. 2. Strong Cl--dependence of Hill activity was observed invariably at all pH values tested (5.5--8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll). 3. In the absence of added Cl- the functionally Cl--depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H2O2 at rates which are 4--12 times faster than the rate of the residual Hill reaction. 4. The Cl--concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl- in equilibrium E . Cl- (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation. 5. The initial phase of the development of inhibition of water oxidation in Cl--depleted chloroplasts during the dark incubation with NH2OH (1/2 H2SO4) is 5 times slower when the incubation medium contains Cl- than when the medium contains NH2OH alone or NH2OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O2 evolution).

Anions↗
PubMed1977

Pathways of silicomolybdate photoreduction and associated photophosphorylation in tobacco chloroplasts.

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (greater than 5 mg/ml) is associated with Photosystem I, since it supports phosphorylation with a P/e2 value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e2 value routinely observed is about 0.2. Bovine serum albumin (1-2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even if the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.

Binding Sites↗