Goro Tamura

Immunochemical characterization of nitrite reductases from spinach leaves, spinach roots and other higher plants

Abstract Rabbit antisera were prepared against native-nitrite reductase and a modified-nitrite reductase isolated from spinach ( Spinacia oleracea ) leaves. Using immunodiffusion and immunoprecipitation techniques, native-nitrite reductase, modified-nitrite reductase and coupling protein (a 24 kDa fragment of the native enzyme) from spinach leaves were characterized. In addition, spinach leaf nitrite reductase was compared to nitrite reductase enzymes isolated from spinach root and from leaves of other higher plants. Native- and modified-nitride reductases appeared to be immunologically very similar. However, antisera against the two preparations were found to inhibit the enzyme reactions to different degrees. The antinative-nitrite reductase produced a single precipitation band against coupling protein, whereas the anti modified-nitrite reductase did not. It was thus concluded that the native- and modified-nitrite reductase of S. oleracea leaves have antigenic determinants in common. Efforts to reconstitute the native-nitrite reductase by addition of coupling protein to modified-nitrite reductase have been unsuccessful to date. Nitrite reductases from spinach leaves and roots appear to be identical proteins, on the basis of their antigenic behavior. Antisera were also used to compare the cross reactivities of nitrite reductase from other higher plants. These tests revealed a high degree of similarity between the S. oleracea and Chenopodium album nitrite reductases. No immunological cross reaction could be detected between nitrite reductase from S. oleracea and the enzymes from Cucurbita pepo, Zea mays, Hordeum vulgare , and Brassica rape .

Isolation and partial characterization of homogeneous glutamate synthase from Spinacia oleracea

Abstract Ferredoxin-dependent glutamate synthase [L-Glutamate: ferredoxin oxidoreductase (transaminating) EC 1.4.7.1] was purified approx. 1100-fold with a yield of 3.8% from spinach leaves using a procedure involving ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose chromatography and Ultrogel AcA 34 gel filtration. The purified enzyme was apparently homogeneous as shown by disc gel electrophoresis and had a specific activity of 35.9 units/mg protein. The molecular weight of the enzyme was estimated to be 180 000 by SDS-polyacrylamide gel electrophoresis. The absorption spectrum of the enzyme in the visible has no prominent maxima or shoulders. The purified enzyme is unstable and is rapidly inactivated during storage event at low temperatures (0–4°C).

Isolation and partial characterization of homogeneous nitrite reductase from etiolated bean shoots (Phaseolus angularis W.F. Wight)

Abstract Methyl viologen-dependent nitrite reductase (EC 1.7.7.1) (NiR) was purified about 1780-fold with a yield of 3.5% from etiolated bean shoots with a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Butyl-Toyopearl chromatography, ferredoxin-Sepharose affinity chromatography and Ultrogel AcA44 gel filtration. The purified enzyme was apparently homogeneous as shown by polyacrylamide disc gel electrophoresis with a specific activity of 53.4 units/mg protein. The molecular weight of the enzyme was estimated to be 100 kilodaltons by gel filtration. Subunit analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two protein bands with a large subunit molecular weight of 64 kilodaltons and a small subunit molecular weight of 35 kilodaltons. The purified enzyme could be stored at −20°C for several weeks without any loss of activity in the presence of 10% glycerol and 10 mM s-mercaptoethanol.

Some properties of ferredoxin-nitrite reductase from green shoots of bean and an immunological comparison with nitrite reductase from roots and etiolated shoots

Abstract Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from green shoots of bean ( Phaseolus angularis ). The isolated enzyme (GR-NiR), having a molecular mass of 68 000, showed 1.4 times higher ferredoxin-dependent activity than methyl viologen-linked activity. The enzyme was homogeneous by polyacrylamide gel electrophoresis (PAGE). In the oxidized form, the enzyme had absorption maxima at 275, 393 (Soret band), 535 and 571 (α band) nm, indicating that siroheme is involved in the catalysis of nitrite reduction. The absorbance ratios, A 393 : A 275 and A 571 : A 393 were 0.26 and 0.32, respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion and immunoelectrophoresis suggested that it was a specific antiserum against GR-NiR. Using the antiserum, immunodiffusion and immunoprecipitation procedures were employed to compare the immunological similarity of NiR from green shoots, etiolated shoots and roots of bean. These tests revealed that the three forms of assimilatory NiR have antigenic determinants in common.

Effect of Sulfur and Nitrogen Nutrition on Derepression of Ferredoxin-Sulfite Reductase in Leek Seedlings

The ferredoxin-sulfite reductase (Fd-SiR; hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1) activities of shoot and root of leek (Allium tuberosum) were increased by sulfate limitation in the early stage of growth. Western blot analysis demonstrated an increased amount of SiRs in root under sulfate limitation, suggesting that SiRs were derepressed. The derepression was observed in shoot when 1.5 mM nitrate was supplied to the plants under sulfate limitation, and clearly in root when 15 mM nitrate was supplied under sulfate limitation. When nitrate was absent from the nutrient solution, the SiR activity in both tissues was very low. Combined with the results of the sulfate- or nitrate-limitation experiments, it is suggested that the degree of the derepression of SiR in both tissue under sulfate limitation is affected by the concentration of nitrate, and further that the mechanism of regulation of the SiR activity is different in each tissue. The decreases in the ratios of the total SiR activities (shoot/root) in the latter stage of seedling growth indicate that root play a very important role in sulfate assimilation.

Purification and Characterization of Ferredoxin-Sulfite Reductases from Leek(Allium tuberosum) Leaves

Ferredoxin-sulfite reductases (Fd-SiRs) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from leek leaves have been purified to homogeneity. The enzymes (SiR 1, SiR 2 and SiR 3) were separated by Mono Q chromatography. The collective molecular mass of the enzymes was estimated to be 65 kDa by gel filtration. In all three cases, subunit analysis by SDS-PAGE yielded a single protein band corresponding to a molecular mass of 64 kDa, indicating that the enzymes each exist as a monomer. In the oxidized forms, SiR 1, SiR 2 and SiR 3 all exhibited nearly identical absorption maxima at 279∼280, 389∼390, 588 and 714 nm, indicating that siroheme is involved in the catalysis of sulfite reduction. On enzymatic properties, SiR 1, SiR 2 and SiR 3 could only react with the physiological electron donor, feriedoxin. The enzymes exhibited different heat stabilities. The pH active curve obtained from SiR 2 was different from the others. Moreover, SiR 1 exhibited a lower Km value for ferredoxin than SiR 2. Although the N-terminal sequence was the same, the results of some enzymatic properties, amino acid analysis, and peptide mapping suggested the presence of the Fd-SiR isozymes in leek leaves.

Ferredoxin isoforms from Chlorella vulgaris (Chlorococcales, Chlorophyceae): Molecular characterization and participation in ferredoxin-dependent enzymes

Using hydrophobic chromatography, Chlorella ferredoxin was separated into three components (Fd I, Fd II and Fd III) in ratios of approximately 3:13:1. The three components differed in isoelectric point, peptide mapping, amino acid composition and N‐terminal sequence. Fd II and Fd III were found to support fairly high rates of cytochrome c reduction by spinach FNR, while Fd I could not support this reaction at all. The highest value of the specificity constant (kcat/Km for NiR was demonstrated for Fd II‐dependent activity; however, the lowest value of kcat/Km for NiR was obtained using Fd II.