An NADP-Glutamate Dehydrogenase from the Green Alga Bryopsis maxima. Purification and Properties

NADP-glutamate dehydrogenase (EC 1.4.1.4 EC] ; NADP-GDH) was purifiedto electrophoretic homogeneity from the multinuclear-unicellulargreen marine alga in Sipho-nales, Bryopsis maxima, and its propertieswere examined. M_r of the undenatured enzyme was 280 kDa, andthe enzyme is thought to be a hexamer of 46 kDa subunit protein.Optimum pHs for the reductive amination and oxidative deaminationwere 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specificactivities with a ratio of 18 :1. Apparent K_m values for 2-oxoglutarate,ammonia, NADPH, glutamate and NADP⁺ were 3.0, 2.2, 0.03, 3.2and 0.01 mM respectively. The enzymochemical characteristicsof the GDH were studied and compared to those of other species.The B. maxima GDH was insensitive to 5 mM Ca²⁺ and to 1 mM EDTAin contrast to higher plant NAD-GDHs. Chemical modificationswith DTNB and pCMBS suggested that cysteine residues are essentialfor the enzymatic activity as in other species GDHs. The GDHwas not affected by 1 mM purine nucleotides, suggesting thatthe enzyme is not allosteric, in contrast to animal NAD(P)-GDHsand fungal NAD-GDHs. (Received August 12, 1996; Accepted January 7, 1997)