1996 Fiscal Year Final Research Report Summary
Purification and characterization of the nitrogen-assimilation enzymes in algae
| Project/Area Number |
07640871
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物生理
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| Research Institution | Toho University |
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Principal Investigator |
OKADA Mitsumasa Toho Univ., Facul.Sci., Professor, 理学部, 教授 (80057629)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Activation / Bryopsis maxima / Glutamate Dehydrogenase / GS / GOGAT / Induction / Localization / Nitrate Reductase / Nitrogen Assimilation |
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| Research Abstract |
(1) Main GDH in algae, NADP-GDH was purified to electrophoretic homogeneiety from the multinuclear unicellular green marine algae, Bryopsis maxima. Molecular mass of the enzyme was 280 kDa comprised of 6 sub-units of 46 kDa. Apparent Km for 2-oxoglutarate, ammonia, NADPH,glutamate and NADP+ were 3.0,2.2,0.03.3.2,0.01mM,respectively. The GDH was insensitive to 5 mM Ca2+ and 1 mM EDTA in contrast to the main GDH in higher plant, NADP-GDH.Chemical modification of with DTNB and pCMBS suggested that cysteine residues are essential for the enzymatic activity. The GDH was not affected by purine nucleotides, suggesting that the enzyme is not allosteric, in contrast to animal and fungal GDHs.
(2) Two major enzyme activities have been detected in chloroplasts and cytosol, with minor one in mitochondria. These three NADP-GDHs were separated by anion-exchange chromatography.
(3) Three new NADP-GDHs were induced while incubation under the continuous light or dark conditions for 5 days in chloroplasts, while intrinsic mitochondrial and cytosolic ones diminished. Four and one NAD-GDHs were induced in chloroplasts and cytosol, respectively, in 30 days.
(4) Localization of other nitrogen assimilation enzymes was also determined : Glutamine synthetase was in chloroplasts and cytosol. Glutamate synthase in chloroplasts. Nitrate reductase in cytosol.
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