Nitrogen metabolism in the macro alga : Induction and localization of isozymes of glutamate dehydrogenase.

研究了绿色大叶藓（Bryopsis maxima）谷氨酸脱氢酶（GDH）的亚细胞定位。采用连续离心和Percoll密度梯度离心两种方法分离完整和纯的叶绿体和线粒体。叶绿体，线粒体和胞质部分的NADP依赖的GDH活性分别估计为64.3%，9.8%和25.9%，仅在叶绿体中观察到NAD依赖的GDH活性。三种细胞器特异性同工酶，叶绿体NADP-GDH 1，胞质/线粒体NADP-GDH 2和胞质/线粒体NADP-GDH 3，被纯化。这些同工酶的分子量估计是相同的（280 kDa）。NADP-GDH 1、NADP-GDH 2和NADP-GDH 3在胺化反应中对NADPH的Km值分别为30、110和34 μ M，对NADH的Km值分别为185、1490和974 μ M，显示出不同的辅因子亲和力。在连续光照和黑暗条件下培养0 ~ 5d，叶绿体中NADP-GDH和NADP-GDH的表达量均被诱导，而线粒体和胞质NADP-GDH的表达量在培养5d后下降到几乎检测不到的水平。它们推导的氨基酸序列有90%的同源性，但有52%的密码子第3位存在同义核苷酸替换。基因组Southern分析表明，这两个基因位于B.maxima染色体上的两个不同位点。因此，证实了B.maxima GDH在蛋白质和基因上都是多元的，并确定了其他氮同化酶的定位。谷氨酰胺合成酶位于叶绿体和细胞质中，谷氨酸合成酶位于叶绿体中，硝酸还原酶位于细胞质中。

Subcellular localization of glutamate dehydrogenase (GDH) wasinvestigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mitochondria were isolated by two methods : successive centrifugation and continuous Percoll density gradient centrifugation. The NADP-dependent GDH activities of the chloroplastic, mitochondrial and cytosolic portions were estimated as 64.3, 9.8, and 25.9 %, respectively, and NAD-dependent GDH activity was observed only in the chloroplasts. Three organelle-specific isozymes, chloroplastic NADP-GDH1, cytosolic/mitochondrial NADP-GDH2 and cytosolic/mitochondrial NADP-GDH3, were purified. The molecular masses of these isozymes were estimated to be the same (280 kDa). The K_m values of NADP-GDHl, NADP-GDH2, and NADP-GDH3 for NADPH in the amination reaction were 30, 110, and 34 muM, respectively, and those for NADH were 185, 1490, and 974 muM, respectively, showing different cofactor affinities. Several NADP-GDHs and one NAD-GDH were induced in the chloroplasts during incubation of the collected thalli in either continuous light or darkness in aerated seawater for 0 to 5 days, while the mitochondrial and cytosolic NADP-GDHs decreased to an almost undetectable level in 5 days.Two distinct DNA fragments (BmF-l and BmF-2) encoding B.maxima GDH were identified and sequenced. They showed 90% homology in their deduced amino acid sequences, whereas, synonymous nucleotide substitution was observed in the third position of 52% codons. Genomic Southern analysis suggests that the two genes are located at two different loci on the B.maxima chromosome. Thus, B.maxima GDH has been confirmed to be multiple in terms of both protein and gene.The localization of other nitrogen-assimilating enzymes was also determined. Glutamine synthetase was located in the chloroplasts and the cytosol ; glutamate synthase was located in the chloroplasts ; and nitrate reductase was located in the cytosol.