Glass T L, Hylemon P B
J Bacteriol. 1980 Mar;141(3):1320-30. doi: 10.1128/jb.141.3.1320-1330.1980.
An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.
通过热处理、硫酸铵分级分离、二乙氨基乙基纤维素、Bio-Gel A 1.5m和羟基磷灰石色谱法，从多形拟杆菌中纯化出一种氧化型烟酰胺腺嘌呤二核苷酸磷酸/氧化型烟酰胺腺嘌呤二核苷酸（NADP⁺/NAD⁺）非特异性L-谷氨酸脱氢酶，其比粗细胞提取物纯化了40倍（NADP⁺或NAD⁺活性）。NADP⁺依赖性和NAD⁺依赖性活性在所有色谱处理中均共洗脱。此外，在每个纯化步骤中，NADP⁺/NAD⁺比活性的比例保持恒定。两种活性在6%非变性聚丙烯酰胺凝胶中也同时迁移。使用Procion RED HE-3B对40倍纯化的酶进行亲和色谱，得到一种同时含有NADP⁺和NAD⁺连接活性的制剂，在十二烷基硫酸钠-聚丙烯酰胺梯度凝胶电泳后显示出一条分子量为48,500的单一蛋白带。该酶的双吡啶核苷酸性质在氧化方向上最为明显。在还原反应中，该酶对还原型NADP的活性比对还原型NAD高30倍。观察到还原型NADP和氯化铵的1/V对1/S曲线呈非线性凹形。盐（0.1M）刺激NADP⁺连接的反应，抑制NAD⁺连接的反应，对还原型NADP依赖性反应影响很小。盐（NADP⁺）的刺激作用是非特异性的，与阴离子或阳离子无关，而NAD⁺连接抑制的程度按I⁻＞Br⁻＞Cl⁻＞F⁻的顺序降低。在其他拟杆菌脱氧核糖核酸同源组的代表性菌株的细胞提取物中也检测到了NADP⁺和NAD⁺谷氨酸脱氢酶活性。
