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Hayakawa T, Yamaya T, Kamachi K, et al. Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures. Plant Physiol. 1992;98(4):1317-22doi: 10.1104/pp.98.4.1317.
Hayakawa, T., Yamaya, T., Kamachi, K., & Ojima, K. (1992). Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures. Plant physiology, 98(4), 1317-22. https://doi.org/10.1104/pp.98.4.1317
Hayakawa, T, et al. "Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures." Plant physiology vol. 98,4 (1992): 1317-22. doi: https://doi.org/10.1104/pp.98.4.1317
Hayakawa T, Yamaya T, Kamachi K, Ojima K. Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures. Plant Physiol. 1992 Apr;98(4):1317-22. doi: 10.1104/pp.98.4.1317. PMID: 16668794; PMCID: PMC1080351.
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Plant Physiol. 1992 Apr;98(4):1317-22. doi: 10.1104/pp.98.4.1317.

Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures.

Plant physiology

T Hayakawa, T Yamaya, K Kamachi, K Ojima

Affiliations

  1. Laboratory of Plant Nutrition, Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981, Japan.

PMID: 16668794 PMCID: PMC1080351 DOI: 10.1104/pp.98.4.1317

Abstract

To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent K(m) values for l-glutamine, 2-oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells.

References

  1. J Biol Chem. 1988 Jul 15;263(20):9651-7 - PubMed
  2. J Biol Chem. 1972 Nov 25;247(22):7407-19 - PubMed
  3. Plant Physiol. 1988 May;87(1):148-54 - PubMed
  4. Plant Cell. 1989 Feb;1(2):241-8 - PubMed
  5. Plant Physiol. 1989 May;90(1):351-8 - PubMed
  6. Plant Physiol. 1982 Apr;69(4):848-52 - PubMed
  7. J Biol Chem. 1991 Feb 5;266(4):2028-35 - PubMed
  8. Plant Mol Biol. 1989 Nov;13(5):611-4 - PubMed
  9. Plant Physiol. 1988 Dec;88(4):1411-7 - PubMed
  10. Plant Physiol. 1988 May;87(1):255-7 - PubMed
  11. Plant Physiol. 1991 Jun;96(2):411-7 - PubMed
  12. Plant Physiol. 1982 Sep;70(3):827-32 - PubMed
  13. Plant Physiol. 1987 Dec;85(4):1036-9 - PubMed
  14. Plant Physiol. 1987 Jul;84(3):578-81 - PubMed
  15. J Bacteriol. 1977 Mar;129(3):1173-82 - PubMed

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