Bryopsis maxima (Chlorophyta) glutamate dehydrogenase: Multiple genes and isozymes

Subcellular localization of glutamate dehydrogenase (GDH) was investigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mi tochondria were isolated by two methods: successive centrifugation and cont inuous Percoll density gradient centrifugation. The NADP-dependent GDH acti vities of the chloroplastic, mitochondrial, and cytosolic portions were est imated as 64.3, 9.8, and 25.9%, respectively, and NAD-dependent GDH activit y was observed only in the chloroplasts. Three organelle-specific isozymes- chloroplastic NADP-GDH1, cytosolic/mitochondrial NADP-GDH2, and cytosolic/m itochondrial NADP-GDH3-were purified. The molecular masses of these isozyme s were estimated to be the same (280 kDa). K-m values of NADP-GDH1, NADP-GD H2, and NADP-GDH3 for NADPH in the amination reaction were 30, 110, and 34 mu M, respectively, and those for NADH were 185, 1490, and 974 mu M, respec tively, showing different cofactor affinities. Several NADP-GDHs and one NA D-GDH were induced in the chloroplasts during incubation of the collected t halli in either continuous light or darkness in aerated seawater for 0 to 5 days, whereas the cytosolic and mitochondrial NADP-GDHs decreased to an al most undetectable level in 5 days. Two distinct DNA fragments (BmF-1 and BmF-2) encoding B. maxima Okamura GDH were identified and sequenced. They showed 90% homology in their deduced a mino acid sequences, whereas synonymous nucleotide substitution was observe d in the third position of 52% Of the codons. Genomic Southern analysis sug gested that the two genes are located at two different loci on the B. maxim a chromosome. Thus, B. maxima GDH has been confirmed to be multiple in term s 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, glu tamate synthase was located in the chloroplasts, and nitrate reductase was located in the cytosol.