Purification and characterization of the nitrogen-assimilation enzymes in algae

(1)从多核单细胞绿色海藻巨型真藓（Bryopsismaxima）中纯化出电泳均一的NADP-GDH。该酶的分子量为280 kDa，由6个46 kDa的亚基组成。2-酮戊二酸、氨、NADPH、谷氨酸和NADP+的表观Km分别为3.0、2.2、0.03、3.2、0.01mM。GDH对5 mM Ca ~（2+）和1 mM EDTA不敏感，而高等植物中的主要GDH NADP-GDH对Ca ~（2+）和EDTA不敏感。GDH不受嘌呤核苷酸的影响，这表明该酶不是变构的，与动物和真菌GDH相反。(2)在叶绿体和细胞质中检测到两种主要的酶活性，在线粒体中检测到较小的酶活性。通过阴离子交换色谱法分离这三种NADP-GDH。(3)三个新的NADP-GDH诱导，而在连续光照或黑暗条件下孵育5天的叶绿体，而内在的线粒体和胞质的减少。在30天内，在叶绿体和细胞质中分别诱导了4个和1个NAD-GDH。(4)其他氮同化酶的定位也被确定：谷氨酰胺合成酶在叶绿体和细胞质中。叶绿体中的谷氨酸合酶。胞质中的硝酸还原酶。

(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.