Rendón J L, Calcagno M, Mendoza-Hernández G, Ondarza R N
Arch Biochem Biophys. 1986 Jul;248(1):215-23. doi: 10.1016/0003-9861(86)90419-4.
Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.
通过一个简单的三步程序，即硫酸铵分级分离、DEAE - 纤维素离子交换色谱和2',5'-ADP - 琼脂糖4B亲和色谱，将极大螺旋藻中的谷胱甘肽还原酶[NAD(P)H:GSSG氧化还原酶，EC 1.6.4.2]纯化了1300倍，达到同质。最适pH为7.0，当磷酸根离子浓度增加时，酶活性显著提高。该酶给出了黄素蛋白典型的吸收光谱，即有三个峰，最大吸光度分别在271、370和460 nm，271 nm处的E1%为23.3。GSSG和NADPH的Km值分别为120±12 μM和3.5±0.9 μM。在测定条件下，辅酶A和谷胱甘肽的混合二硫键也被该酶还原，但该酶对该底物的亲和力非常低（Km为3.3 mM）。该酶对NADPH具有特异性。天然酶在4℃时的等电点为4.35，氨基酸组成与先前从其他来源报道的非常相似。变性条件下亚基的分子量为47,000±1200。通过多种分子量测定技术对纯酶的分析表明，在pH 7.0时，该酶主要以四聚体形式存在，并与少量二聚体部分处于平衡状态。在碱性pH（9.5）或6 M尿素中可实现解离成二聚体。然而，中性pH下的平衡不受NADPH或二硫键还原试剂的影响。寡聚酶的Mr和S20,w估计分别为177,000±14,000和8.49±0.5；二聚体的分别为99,800±7000和5.96±0.4。低浓度的尿素增加了酶活性，但这种增加不是由于两种形式比例的变化。
