In the past Osamu Hayaishi has collaborated on articles with Hiroshi Okamoto and Ryotaro Yoshida. One of their most recent publications is Enzymic oxidation of reduced α-nicotinamide adenine dinucleotide☆. Which was published in journal Archives of Biochemistry and Biophysics.

More information about Osamu Hayaishi research including statistics on their citations can be found on their Copernicus Academic profile page.

Osamu Hayaishi's Articles: (73)

Enzymic oxidation of reduced α-nicotinamide adenine dinucleotide☆

AbstractReduced α-nicotinamide adenine dinucleotide (α-NADH) is oxidized at about one fifth of the rate of reduced β-nicotinamide adenine dinucleotide by rat liver mitochondria that have been treated with a hypotonic medium. Essentially no oxidation is observed with intact mitochondria. Oxidation is almost completely inhibited by 1 mm cyanide, but is insensitive to 1 μm antimycin A, 1 μm rotenone, and 2 mm anytal.Furthermore, enzyme activities have been detected that catalyze the transfer of electrons from α-NADH to cytochrome c and to 2,6-dichlorophenolindophenol and are seemingly localized in the microsomal and soluble fractions, respectively.

Specific induction of indoleamine 2,3-dioxygenase by bacterial lipopolysaccharide in the mouse lung☆

AbstractIndoleamine 2,3-dioxygenase activity in the supernatant fractions (30,000g, 30 min) from various tissues of mice increased almost linearly after a single intraperitoneal administration of bacterial lipopolysaccharide (5 to 20 μg/mouse). The most prominent effect was observed in the lung, where both specific and total enzyme activities increased 40 to 80-fold during the first 24 h. Significant (10- to 20-fold) stimulation was also observed in the seminal vesicle, coagulating gland, colon, and caecum, and severalfold in the trachea, stomach, heart, small intestine, and spleen. Lipid A fraction, the biologically active unit in the lipopolysaccharide complex, was as active as the lipopolysaccharide preparations from either Escherichia coli or Salmonella S and R mutant strains, whereas the polysaccharide fraction was inactive under identical experimental conditions. When mice were pretreated with a series of daily injections of bacterial lipopolysaccharide, enzyme induction was no longer evident, indicating that tolerance to this agent had developed and that enzyme induction was caused by lipopolysaccharide but not by possible contaminants in the preparations. The enzyme activities from normal and lipopolysaccharide-treated mice were exclusively found in the soluble fractions of mouse lung homogenates. Other enzyme activities in the lung such as lysosomal (acid phosphatase), microsomal (prostaglandin cyclooxygenase), mitochondrial (monoamine oxidase and superoxide dismutase), and soluble enzyme activities (lipooxygenase and superoxide dismutase) were not significantly altered by this treatment. This increase in the enzyme activity with the lipopolysaccharide treatment was abolished with a simultaneous administration of cycloheximide or actinomycin D, and an immunological analysis with antibody for mouse enzyme (rabbit IgG) demonstrated that the observed increment of the enzyme activity was essentially due to an increase in the enzyme protein.

Inhibition of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase by β-carboline and indole derivatives☆

Abstractβ-Carboline derivatives inhibited both indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase activities from various sources. Among them, norharman is most potent for both enzymes from mammalian sources. Kinetic studies revealed that norharman is uncompetitive (Ki = 0.12 mm) with l-tryptophan for rabbit intestinal indoleamine 2,3-dioxygenase, and linearly competitive (Ki = 0.29 mm) with l-tryptophan for mouse liver tryptophan 2,3-dioxygenase. In addition, some β-carbolines selectively inhibited one enzyme or the other. Pseudomonad tryptophan 2,3-dioxygenase was inhibited by a different spectrum of β-carbolines. Such a selective inhibition by the structure of substrate analogs is more evident by the use of indole derivatives. Indole-3-acetamide, indole-3-acetonitrile and indole-3-acrylic acid exhibited a potent inhibition for mammalian tryptophan 2,3-dioxygenase, while they moderately inhibited the pseudomonad enzyme. However, they showed no inhibition for indoleamine 2,3-dioxygenase. These results suggest the difference of the structures of the active sites among these enzymes from various sources.

Purification and characterization of prostaglandin F synthase from bovine liver☆

AbstractProstaglandin D2 11-ketoreductase activity of bovine liver was purified 340-fold to apparent homogeneity. The purified enzyme was a monomeric protein with a molecular weight of about 36 kDa, and had a broad substrate specificity for prostaglandins D1, D2, D3 and H2 and various carbonyl compounds (e.g., phenanthrenequinone and nitrobenzaldehyde, etc.). Prostaglandin D2 was reduced to 9α,11β-prostaglandin F2 and prostaglandin H2 to prostaglandin F2α with NADPH as a cofactor. Phenanthrenequinone competitively inhibited the reduction of prostaglandin D2, while it did not inhibit that of prostaglandin H2. Moreover, chloride ion stimulated the reduction of prostaglandin D2 and carbonyl compounds, while it had no effect on that of prostaglandin H2. Besides, the enzyme was inhibited by flavonoids (e.g., quercetin) that inhibit carbonyl reductase, but was not inhibited by barbital and sorbinil, which are the inhibitors of aldehyde and aldose reductases, respectively. These results indicate that the bovine liver enzyme has two different active sites, i.e., one for prostaglandin D2 and carbonyl compounds and the other for prostaglandin H2, and appears to be a kind of carbonyl reductase like bovine lung prostaglandin F synthase (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O., 1985, J. Biol. Chem. 260, 7035–7041). However, the bovine liver enzyme was different from prostaglandin F synthase of bovine lung with regard to the Km value for prostaglandin D2 (10 μm for the liver enzyme and 120 μm for the lung enzyme), the sensitivity to chloride ion (threefold greater activation for the liver enzyme) and the inhibition by CuSO4 and HgCl2 (two orders of magnitude more resistant in the case of the liver enzyme). These results suggest that the bovine liver enzyme is a subtype of bovine lung prostaglandin F synthase.

Purification and some properties of protocatechuate 4,5-dioxygenase☆

Abstract1.1. Protocatechuate 4,5-dioxygenase (protocatechuate:oxygen 4,5-oxidoreductase, EC was purified from a pseudomonad to an almost homogeneous state. The most purified preparation had a specific activity of 160 μmoles/min per mg of protein at 24° and the molecular weight was estimated to be approx. 150 000. The enzyme contained one atom of iron per mole of the enzyme protein.2.2. The enzyme was very unstable and was easily inactivated during storage either at 0° or at room temperature. The inactivation of the enzyme was counteracted by the presence of 10% ethanol. The enzyme was also inactivated in the presence of oxidizing agents such as H2O2 even in the presence of 10% ethanol. The inactivated enzyme was partly reactivated by the incubation with both Fe2+ and ascorbic acid.3.3. Rapid inactivation of the enzyme was observed during the catalysis. The inactivation was found to be due simply to the removal of the iron from the enzyme protein, since the inactivated enzyme was fully reactivated by the addition of Fe2+ alone. These results together with the fact that the enzyme was not inactivated by incubation with chelating agents suggest that the interaction between the iron and the enzyme protein is altered during the catalysis.

Adenyl cyclase of Brevibacterium liquefaciens

AbstractAdenyl cyclase has been purified about 100-fold from cell-free extracts of Brevibacterium liquefaciens. The purified enzyme preparation catalyzed the conversion of ATP (or dATP) to cyclic 3′,5′-AMP (or cyclic 3′,5′-dAMP) and pyrophosphate. In addition to Mg2+, pyruvate or another α-keto-monocarboxylic acid was required for the reaction. None of the other nucleoside triphosphates tested served as substrate.

New degradative routes of 5-hydroxytryptophan and serotonin by intestinal tryptophan 2,3-dioxygenase☆

AbstractA highly purified preparation of tryptophan 2,3-dioxygenase from rabbit intestine was found to catalyze the oxygenative ring cleavage of 5-hydroxytryptophan and serotonin. The products of these enzymic reactions were susceptible to the action of formamidase, as a consequence of which 5-hydroxykynurenine and 5-hydroxykynurenamine were isolated from the reaction mixtures and identified. The initial products were presumed, therefore, to be 5-hydroxyformylkynurenine and 5-hydroxyformylkynurenamine, respectively. Several lines of evidence indicated that the cleavage of tryptophan, 5-hydroxytryptophan and serotonin occurred by the action of a single protein, namely intestinal tryptophan 2,3-dioxygenase.

Studies on a possible reaction intermediate of p-hydroxyphenylpyruvate dioxygenase

AbstractA quinol has been proposed to be an intermediate of the p-hydroxyphenylpyruvate dioxygenase-catalyzed reaction. However, using a highly purified enzyme from bovine liver and chemically synthesized quinol, no significant formation of homogentisate, the product of the enzymic reaction, from the quinol was observed under any conditions tested. Furthermore, the quinol showed no appreciable inhibition on the enzymic activity. In light of these findings, the reaction mechanism of the enzyme is discussed.

ADP-ribosylation of nuclear protein A24☆

AbstractNuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [Goldknopf and Busch (1977) Proc. Natl. Acad. Sci. USA74, 864–868], is found to be ADP-ribosylated in isolated rat liver nuclei.

Superoxide anion as a cofactor of dopamine-β-hydroxylase

AbstractSuperoxide anion can serve a reducing agent for tyramine hydroxylation by dopamine-β-hydroxylase. Stable O2• solutions were obtained by dissolving KO2 in dry dimethylsulfoxide and infused into buffered solutions of tyramine and dopamine-β-hydroxylase at constant rate. The reaction requires molecular oxygen, but differs from the ascorbate dependent hydroxylation in its alkaline pH optimum value (pH 7.5) and its low rate (9 nmol octopamine formed/min/mg of protein). In absence of tyramine O2• does not produce a stable reduced form of the enzyme.

Snake venom phosphodiesterase: Simple purification with Blue Sepharose and its application to poly(ADP-ribose) study☆

AbstractA rapid method of purifying snake venom phosphodiesterase has been developed using Blue Sepharose or blue dextran/Sepharose as an affinity adsorbent. A sixty-fold purification of the enzyme from commercial preparations is achieved in a single step with a yield of 60%. The purified enzyme preparation is essentially free from phosphatase activities and exhibits a major protein band on SDS-polyacrylamide gel electrophoresis. Chain length analysis of poly(ADP-ribose) exemplifies the usefulness of this technique.

Prostaglandin D2, a potential antineoplastic agent☆

AbstractCytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D2 (PGD2) was found most active. PGD2 exerted a dose dependent inhibition of L1210 cell growth over 3.6 μM. At 14.3 μM growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC50 value of PGD2 on L1210 cell growth was calculated to be 6.9 μM (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD2 was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA2 showed a comparable, and PGE2 a less but significant growth inhibitory activity, while PGB2, PGF2α and PGI2 had no such effects on cell proliferation at 14.3 μM concentration. These results suggest a potential antineoplastic activity of PGD2.

Involvement of GTP-regulatory protein in brain prostaglandin E2 receptor and separation of the two components

AbstractThe specific binding protein for prostaglandin (PG) E2 solubilized from porcine brain was sensitive to guanine nucleotides. GTP inhibited the association and enhanced the dissociation of the specific [3H]PGE2 binding. Scatchard analyses showed that GTP (10 μM) decreased the binding affinity more than 3-fold without major change in the number of binding site. Gel filtration separated the binding site from GTP-regulatory component (N). The separated binding protein had a reduced affinity to PGE2 and lost its sensitivity to GTP. The addition of the separated N restored its responsiveness to GTP, and also increased the binding affinity to the original level. These results provide direct evidence for the molecular interaction between the PGE2 binding protein and N in the brain.

Arachidonic acid stimulates phosphoinositide metabolism and catecholamine release from bovine adrenal chromaffin cells

AbstractArachidonic acid (AA) evoked a dose-dependent increase in the accumulation of inositol phosphates in cultured bovine adrenal chromaffin cells, and this effect was specific for AA. AA also induced a rise in [Ca2+]i, but this rise was markedly reduced by removal of extracellular Ca2+. AA-induced accumulation of inositol phosphates was absolutely dependent on extracellular Ca2+, and nicardipine and nifedine partially reduced it but verapamil had no effect. Moreover, AA dose-dependently stimulated catecholamine release from chromaffin cells in the presence of ouabain, and this effect was specific for AA. AA-induced catecholamine release in the presence of ouabain was also inhibited by nicardipine and nifedipine but not by verapamil. Furthermore, the phospholipase C inhibitor neomycin inhibited the release. These results taken together suggest that AA stimulates catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism in a Ca2+-dependent manner.

Research reportSuppression of sleep by prostaglandin synthesis inhibitors in unrestrained rats

AbstractSleep-suppressive activity of prostaglandin synthesis inhibitors, diclofenac sodium (DF) and indomethacin (IM), was examined in unrestrained male rats. An intraperitoneal injection of 5 mg/kg DF and 10 mg/kg IM at an early phase of the light period transiently decreased slow wave sleep (SWS) and paradoxical sleep (PS) to 30–62% and 0–38%, respectively, of the control level in the first hour. An intravenous infusion of 0.4 mg DF or 0.4 mg IM or an intracerebroventricular infusion of 0.04 mg DF continuously during a 10-h diurnal period resulted in a significant decrease in SWS and PS by 9–17% an 17–21%, respectively, from the baseline value in the 12-h light period. The DF infusion was accompanied by a rebound rise in the nocturnal SWS and PS and the subsequent diurnal PS. The results indicate that the depletion of prostaglandin(s) in the brain is responsible for the DF- and IM-induced suppression of sleep.

Research reportAwaking effect of prostaglandin E2 in freely moving rats

AbstractThe awaking effect of prostaglandin (PG) E2 was further examined in a long-term bioassay system. PGE2 in saline solution was infused between 11.00 and 17.00 h at 0.1, 1, 10, and 100 pmol/min (infusion volume 10 μl/h) into the third cerebral ventricle of freely moving rats. These rats were otherwise infused with saline continuously and exhibited a circadian cycle, spending 70% of the daytime and 37% of the night in sleep. In the rats that received PGE2 infusion at 1, 10 and 100 pmol/min, slow wave sleep (SWS) decreased to 84%, 69% and 71% and paradoxical sleep (PS) to 85%, 37% and 40% of the paired controls. Thus, the effect of PGE2 was not specific to either SWS or PS. No effects were observed in the rats that received PGE2 at 0.1 pmol/min. After PGE2 infusion at 10 and 100 pmol/min, marked rebounds of both SWS and PS occurred during the night. SWS reduction by PGE2 was due to the shortened duration of SWS episodes, while SWS increase in the rebound phase was due to the increased number of episodes. PS reduction was due to both the shortened duration and decreased number of PS episodes and PS rebound was due to both the prolonged duration and increased number of episodes. The circadian sleep-wake cycle returned to the baseline on the first or second recovery day after PGE2 infusion. Sleep reduction by PGE2 was accompanied by elevation of the brain temperature and rebound increase of sleep occurred with the fall of the brain temperature. On the basis of these findings, we conclude that PGE2 has a central awaking effect without irreversible damage to the sleep-wake regulating mechanism and that the magnitude of the effect depends on the amount of PGE2 at the site of action in the brain.

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