Toshiyuki Ohnishi

One-electron-transfer reactions in biochemical systems II. The reaction of free radicals formed in the enzymic oxidation

Free radicals formed in the enzymic oxidation are extremely reactive and act as strong reductants or oxidants. The rate constants of the reaction of free radicals with cytochromes are measured directly using an electron spin resonance (ESR) spectrometer and a sensitive spectrophotometer, both equipped with a flow apparatus. 2-Methyl-1,4-naphthosemiquinone (MKH), for instance, is a very active reductant and reduces cytochrome c and cytochrome b5. THe rate constants are about 3·108 M−1. sec−1. MKH is also found to react with molecular oxygen at a rate constant of roughly 5·106 M−1·sec−1. The rate constants of cytochrome c reduction by monodehydroascorbate and p-benzosemiquinone are measured directly as 4.0·104 and 2.5·106 M−1. sec−1, respectively. On the other hand, free radicals derived from p-cresol and chlorpromazine act as effective one-electron oxidants. The one-electron transfer from donor molecules to these free radicals is confirmed by means of ESR spectroscopy.

One-electron-transfer reactions in biochemical systems I. Kinetic analysis of the oxidation-reduction equilibrium between quinol-quinone and ferro-ferricytochrome c

Abstract At neutral pH the reduction of cytochrome c by quinol (hydroquinone) is greatly accelerated in the presence of quinone. This acceleration is ascribed to the formation of p -benzosemiquinone. The reduction of cytochrome c by the semiquinone is fast and plays an important role in the oxidation-reduction equilibrium between ferro-ferricytochrome c and quinol-quinone. Kinetic studies using an electron spin resonance technique indicate the following reaction mechanism: The equilibrium constant, K ′ for the overall reaction, is equal to ( k f ′/ k d ) ( k r / k o ) 2 at neutral pH. The values calculated from the kinetics are in good agreement with those which have been obtained electrometrically.

On the active site of myosin A-adenosine triphosphatase IV. Properties of binding of trinitrobenzenesulfonate and p-chloromercuribenzoate to myosin a

Abstract The addition of ATP or PP i retarded conspicuously the rate of specific binding of trinitrobenzenesulfonate to one mole of lysine residue in 2.1·10 5 g of myosin A. The rate of specific binding of trinitrobenzenesulfonate to myosin A was increased in 4M LiBr which melts almost completely the helical structure of myosin A. The rate was also remarkably influenced by the treatment of myosin A with 1.5 M LiBr which inactivated ATPase (ATP phosphohydrolase, EC 3.6.1.3) without changing significantly the helical content of the myosin A molecule as a whole. Furthermore, it was demonstrated that actomyosin reconstituted from myosin A treated with p -chloromercuribenzoate and β-mercaptoethanol does not show a clearing response on addition of high concentrations of ATP and its ATPase activity is not inhibited by the substrate or by EDTA. The p -chloromercuribenzotae added was completely removed from myosin A by the further addition of excess β-mercaptoethanol and the optical rotatory dispersion of myosin A was insignificantly altered by the treatment with p -chloromercuribenzoate and β-mercaptoethanol.

An experimental approach to study the biosynthesis of brominated metabolites by the red algal genus Laurencia

The production of labeled brominated metabolites with radioactive (82)Br in Laurencia species was investigated as part of a study of the biosynthesis of halogenated metabolites from species belonging to the red algal genus Laurencia (Rhodomelaceae, Ceramiales). Radiobromide [(82)Br], thin-layer chromatography (TLC), and TLC-autoradioluminography (ARLG) were used. When cultured in artificial seawater medium (ASP(12)NTA including Na(82)Br) under 16:8 h light:dark (LD) illumination cycles for 24 h, each of the strains of Laurencia, Laurencia japonensis Abe et Masuda, Laurencia nipponica Yamada (laurencin-producing race and laureatin-producing race), and Laurencia okamurae Yamada, produced species- (or race-) specific (82)Br-containing metabolites. In the case of the laurencin-producing race of L. nipponica, laurencin and deacetyllaurencin were found to be produced in approximately 1:1 ratio, though laurencin is the major metabolite in the wild sample. Furthermore, when cultured in the dark, the production rates of brominated metabolites in Laurencia spp. were found to be diminished. The present study strongly indicates that the use of radiobromine [(82)Br] in combination with the TLC-ARLG method is an effective approach for investigating the biosynthesis of brominated metabolites in Laurencia.

The oxidation-reduction characteristics of cytochrome b5 in hen liver microsomes

Abstract Hen liver microsomes contained 0.20 nmol of cytochromeb5 per mg of protein. Upon addition of NADH about 95% cytochrome b5 was reduced very fast with a rate constant of 206 s−1 When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b5 in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 108 m −1 s−1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b5. To explain the results, therefore, the reverse electron flow from cytochrome b5 to the flavin coenzyme in microsomes was assumed. By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b5 reductase by NADH is rate-limiting in the NADH-cytochrome b5 and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μ m and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μ m .

Tritium Generation and Large Excess Heat Evolution by Electrolysis in Light and Heavy Water-Potassium Carbonate Solutions with Nickel Electrodes

The generation of tritium was quantitatively measured in an electrolytic cell with a nickel cathode and a platinum anode in potassium carbonate-light and heavy water solutions. Simultaneously, the evolution of a large amount of excess heat (70 to 170{degrees} for the input power) was observed during electrolysis of these solutions. The tritium generation by electrolysis provides some of the most conclusive evidence for so-called cold fusion, along with the calcium generation described in a previous paper. On the basis of the current experiments and the knowledge of the knetics of a hydrogen evolution reaction in an alkaline solution, the nuclear reactions taking place are worth mentioning. 11 refs., 1 fig., 2 tabs.

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin.

CD23, a low‐affinity IgE receptor, is a type II transmembrane protein having a C‐type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co‐exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L‐KT9 cells (an Epstein‐Barr virus (EBV)‐transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti‐CD23 and anti‐Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross‐linking of CD23 with anti‐CD23 and its second antibody as compared with the case of stimulation by anti‐CD23 alone. The cross‐linking of CD23 with anti‐CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L‐KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.

Observation of Tritium Distribution in Pure Vanadium and V-5 mol%Fe Alloy by Tritium Radioluminography

ABSTRACT Tritium radioluminography was applied to pure vanadium and V-5 mol%Fe alloy to observe the tritium distribution and to evaluate the local tritium concentration in them. It was demonstrated that the tritium distribution at a microscopic area in the specimens was quantitatively and graphically displayed. In the pure vanadium specimen, the local tritium concentration was about three times different depending on the crystal orientation of the grains. The tritium radioactivity of the grains with (001) and (111) orientation are 1 Bq/mm2 and 0.4 Bq/mm2, respectively. These values correspond to the tritium concentration of 15 mol ppb and 6 mol ppb. The difference of the local tritium concentration was attributed to the variety of the morphology of precipitated hydride depending on the crystal orientation of the grains. For the radioactivity recorded in the imaging plate, the component of the X-rays generated from tritium in the specimen was only 2%, i.e., most of the intensity was attributed to the β-rays. In the V-Fe alloy specimen, it was shown that the tritium distribution correlates with iron segregation formed during solidification after the arc melting. The cross sectional observation showed that the local tritium concentration in equilibrium distribution depends on the local iron concentration in the specimen. The local tritium concentration gradually decreases from 115 mol ppb to 70 mol ppb as the iron concentration at the iron segregated region increases from 3 mol% to 4.5 mol%.

Utilization of a Computer Using Spreadsheet Software for Radiation Protection Management.

We have developed a personal computer system using a commercial spreadsheet software. This system is applicable to management of radioactive wastes and measurement at pertinent points (surface contamination and radiation dose) . As the result of our experience, we have found that the spreadsheet software is quite satisfactory for radiation protection management and that it has advantages in some aspects against other so f twares by programming language.