Inokuchi R, Itagaki T, Wiskich J T, Nakayama K, Okada M
Department of Biomolecular Science, Toho University, Chiba, Japan.
Plant Cell Physiol. 1997 Mar;38(3):327-35. doi: 10.1093/oxfordjournals.pcp.a029170.
NADP-glutamate dehydrogenase (EC 1.4.1.4:NADP-GDH) was purified to electrophoretic homogeneity from the multinuclear-unicellular green marine alga in Siphomales, Bryopsis maxima, and its properties were examined. M(r) of the undenatured enzyme was 280 kDa, and the enzyme is thought to be a hexamer of 46 kDa subunit protein. Optimum pHs for the reductive amination and oxidative deamination were 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specific activities 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, 3.2 and 0.01 mM respectively. The enzymochemical characteristics of the GDH were studied and compared to those of other species. The B. maxima GDH was insensitive to 5 mM Ca(2+) and to 1 mM EDTA in contrast to higher plant NAD-GDHs. Chemical 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.
从管藻目多核单细胞绿色海洋藻类巨藻中纯化出了NADP - 谷氨酸脱氢酶（EC 1.4.1.4：NADP - GDH），并对其性质进行了研究。未变性酶的分子量为280 kDa，该酶被认为是由46 kDa亚基蛋白组成的六聚体。还原胺化和氧化脱氨的最适pH分别为7.5和8.2 - 9.0。该酶表现出NADPH/NADH特异性活性，其比值为18:1。2 - 酮戊二酸、氨、NADPH、谷氨酸和NADP⁺的表观K(m)值分别为3.0、2.2、0.03、3.2和0.01 mM。对GDH的酶化学特性进行了研究，并与其他物种的进行了比较。与高等植物NAD - GDHs不同，巨藻GDH对5 mM Ca(2+)和1 mM EDTA不敏感。用DTNB和pCMBS进行的化学修饰表明，与其他物种的GDHs一样，半胱氨酸残基对酶活性至关重要。GDH不受1 mM嘌呤核苷酸的影响，这表明与动物NAD(P) - GDHs和真菌NAD - GDHs不同，该酶不是别构酶。
