Grantham W C, Brown A T
Arch Oral Biol. 1983;28(4):327-38. doi: 10.1016/0003-9969(83)90075-4.
Capnocytophaga ochracea strain 25 was originally isolated from a patient with severe juvenile periodontitis. An NAD-specific glutamate dehydrogenase (GDH) (EC 1.4.1.2.) was found in cell-free extracts from this organism. The NADH-dependent reductive, or ammonia-assimilating activity (NADH-GDH), of the enzyme was 8-10-fold higher than its NAD-dependent oxidative, or ammonia-releasing activity (NAD-GDH), suggesting that the primary physiological role of the GDH is ammonia-fixing. Capnocytophaga ochracea GDH was purified approximately 39-fold by a rapid, single-step purification procedure using DEAE-cellulose (DE52) ion-exchange column chromatography which gave 90 per cent recovery of total enzyme units. Paper chromatography of an NADH-GDH assay mixture containing the partially purified enzyme showed that glutamate was, indeed, a product of the ammonia-assimilating reaction. The pH optimum for the NAD-GDH reaction was 9.0; that for the NADH-GDH reaction was 7.5. Although a number of mono- and divalent cations were tested, none had a large effect on either NAD-GDH or NADH-GDH activity. The NAD-GDH reaction showed a hyperbolic kinetic response to glutamate and NAD and the Km values for glutamate and NAD were 2.44 and 0.083 mM respectively. The kinetic response of the NADH-GDH reaction to NADH, alpha-ketoglutarate and ammonium chloride also obeyed Michaelis-Menten kinetics and their respective Km values were 0.069, 1.44 and 3.33 mM. Of a number of biologically-active compounds tested for their ability to modulate GDH activity, only ADP and NAD exerted much effect. The NADH-GDH activity showed a negative hyperbolic kinetic response to both ADP and NAD and Dixon plot-analysis of the NAD and ADP saturation data gave Ki values for ADP and NAD of 4.0 and 0.46 mM respectively. Both NAD and ADP appeared to exert their negative effects on NADH-GDH activity by completely inhibiting the binding of the reduced coenzyme, NADH, to the enzyme.
赭黄纤毛菌25株最初是从一名重度青少年牙周炎患者中分离出来的。在该菌株的无细胞提取物中发现了一种NAD特异性谷氨酸脱氢酶（GDH）（EC 1.4.1.2.）。该酶的NADH依赖性还原或氨同化活性（NADH-GDH）比其NAD依赖性氧化或氨释放活性（NAD-GDH）高8至10倍，这表明GDH的主要生理作用是固定氨。使用DEAE-纤维素（DE52）离子交换柱色谱法通过快速单步纯化程序将赭黄纤毛菌GDH纯化了约39倍，总酶单位回收率达90%。对含有部分纯化酶的NADH-GDH测定混合物进行纸色谱分析表明，谷氨酸确实是氨同化反应的产物。NAD-GDH反应的最适pH为9.0；NADH-GDH反应的最适pH为7.5。尽管测试了多种单价和二价阳离子，但它们对NAD-GDH或NADH-GDH活性均无显著影响。NAD-GDH反应对谷氨酸和NAD呈现双曲线动力学响应，谷氨酸和NAD的Km值分别为2.44和0.083 mM。NADH-GDH反应对NADH、α-酮戊二酸和氯化铵的动力学响应也符合米氏动力学，它们各自的Km值分别为0.069、1.44和3.33 mM。在测试的多种具有调节GDH活性能力的生物活性化合物中，只有ADP和NAD有较大影响。NADH-GDH活性对ADP和NAD均呈现负双曲线动力学响应，对NAD和ADP饱和数据进行狄克逊图分析得出ADP和NAD的Ki值分别为4.0和0.46 mM。NAD和ADP似乎都是通过完全抑制还原辅酶NADH与酶的结合来对NADH-GDH活性产生负面影响。
