Laboratory of Plant Nutrition, Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981, Japan.
Plant Physiol. 1992 Apr;98(4):1317-22. doi: 10.1104/pp.98.4.1317.
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.
为了获得 NADH 依赖性谷氨酸合酶（NADH-GOGAT；EC 1.4.1.14）的单克隆抗体，我们使用柱层析法，使用 Butyl Toyopearl 650M、Sephacryl S-300、Blue Sepharose CL-6B 和 Butyl Toyopearl 650S 从培养的水稻细胞（Oryza sativa）中纯化该酶至均一性。在纯化的最后阶段，酶的比活为每分钟每毫克蛋白形成 9.8 微摩尔谷氨酸。收率为 6.1%，纯化倍数为 815 倍。变性凝胶电泳分析显示，单一多肽的表观分子量为 196,000，与天然蛋白估计的 194,000 值相似。对 l-谷氨酰胺、2-氧戊二酸和 NADH 的表观 K(m)值分别为 811、76 和 3.0 微摩尔。NADPH 和 l-天冬酰胺均不能替代 NADH 和 l-谷氨酰胺。该酶的吸收最大值在 273、373 和 440 纳米处，在 475 纳米处有一个肩峰，表明水稻 NADH-GOGAT 是一种黄素蛋白。从水稻细胞中纯化的 NADH-GOGAT 针对单克隆抗体的获得是高等植物中该酶的首例。免疫分析表明，针对水稻细胞 NADH-GOGAT 的抗体仅与细胞提取物中的酶发生反应。抗-NADH-GOGAT 抗体不识别从水稻叶片中纯化的铁氧还蛋白-GOGAT，同样，抗水稻叶片铁氧还蛋白-GOGAT 抗体也不与从培养的水稻细胞中纯化的 NADH-GOGAT 反应。
