|Budget Amount *help
¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1998 : ¥1,100,000 (Direct Cost : ¥1,100,000)
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.