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. 1992 Apr;98(4):1317–1322. doi: 10.1104/pp.98.4.1317

Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures 1

Toshihiko Hayakawa 1,2, Tomoyuki Yamaya 1,2, Kazunari Kamachi 1,2, Kunihiko Ojima 1,2
PMCID: PMC1080351  PMID: 16668794

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 Km 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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