Duhaiman A S, Rabbani N, AlJafari A A, Alhomida A S
Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia.
Biochem Biophys Res Commun. 1995 Oct 13;215(2):632-40. doi: 10.1006/bbrc.1995.2511.
zeta-crystallin a novel NADPH: quinone oxidoreductase was purified from the cortex of the camel (Camelus dromedarius) lens to homogeneity by Sepharose CL-6B gel filtration column and 2', 5' ADP-Sepharose 4B affinity column chromatography in the presence of dithiothreitol. The purified zeta-crystallin has a molecular weight of 140 kDa, as determined by Superose 12 gel filtration column. SDS-PAGE showed a single polypeptide band of molecular weight 35 kDa, suggesting that the native enzyme is composed of four identical subunits. The isoelectric point of the enzyme was 7.6 on native polyacrylamide gel. The enzyme was purified 20-fold over homogenate with a specific activity of 26.0 Units/mg protein, and an overall recovery of 53%. This enzyme was NADPH specific and followed Michaelis-Menten kinetics. Km values for the reduction of 9,10-phenanthroquinone and oxidation of NADPH were 17 microM and 6.9 microM, respectively, at pH 7.8. The Vmax values of the enzyme for 9,10 phenanthroquinone and NADPH were 32 mumole min-1, mg-1 protein and 22.7 mumole min-1 mg-1 protein, respectively.
ζ-晶体蛋白是一种新型的NADPH：醌氧化还原酶，在二硫苏糖醇存在的情况下，通过琼脂糖CL-6B凝胶过滤柱和2',5'-ADP-琼脂糖4B亲和柱色谱法从骆驼（单峰驼）晶状体皮质中纯化至同质。通过Superose 12凝胶过滤柱测定，纯化后的ζ-晶体蛋白分子量为140 kDa。SDS-PAGE显示出一条分子量为35 kDa的单一条带，表明天然酶由四个相同的亚基组成。在天然聚丙烯酰胺凝胶上，该酶的等电点为7.6。该酶比匀浆纯化了20倍，比活性为26.0单位/毫克蛋白质，总回收率为53%。这种酶对NADPH具有特异性，并遵循米氏动力学。在pH 7.8时，还原9,10-菲醌和氧化NADPH的Km值分别为17 μM和6.9 μM。该酶对9,10-菲醌和NADPH的Vmax值分别为32微摩尔·分钟-1·毫克-1蛋白质和22.7微摩尔·分钟-1·毫克-1蛋白质。
