AN NADP-GLUTAMATE DEHYDROGENASE FROM THE GREEN-ALGA BRYOPSIS-MAXIMA -PURIFICATION AND PROPERTIES

NADP-glutamate dehydrogenase (EC 1.4.1.4; NADP-GDH) was purified to el ectrophoretic homogeneity from the multinuclear-unicellular green mari ne alga in Siphonales, Bryopsis maxima, and its properties were examin ed, M(r) of the undenatured enzyme was 280kDa, and the enzyme is thoug ht to be a hexamer of 46 kDa subunit protein, Optimum pHs for the redu ctive amination and oxidative deamination were 7.5 and 8.2-9.0 respect ively, The enzyme displayed NADPH/NADH-specific activities with a rati o of 18 : 1. Apparent K-m, values for 2-oxoglutarate, ammonia, NADPH, glutamate and NADP(+) were 3.0, 2.2, 0.03, 3.2 and 0.01 mM respectivel y, The enzymochemical characteristics of the GDH were studied and comp ared to those of other species, The B. maxima GDH was insensitive to 5 mM Ca2+ and to 1 mM EDTA in contrast to higher plant NAD-GDHs, Chemic al modifications with DTNB and pCMBS suggested that cysteine residues are essential for the enzymatic activity as in other species GDHs, The GDH was not affected by 1 mM purine nucleotides, suggesting that the enzyme is not allosteric, in contrast to animal NAD(P)-GDHs and fungal NAD-GDHs.