Ulusu N N, Kus M S, Acan N L, Tezcan E F
Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Int J Biochem Cell Biol. 1999 Jul;31(7):787-96. doi: 10.1016/s1357-2725(99)00019-9.
This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.
本文描述了一种从牛晶状体中纯化葡萄糖-6-磷酸脱氢酶的简单快速方法，以及对该酶的动力学行为和一些特性的分析。纯化过程包括两个步骤，即2',5'-ADP-琼脂糖4B亲和层析和DEAE琼脂糖快速流动离子交换层析，整个过程耗时两个工作日。获得的酶产率为13.7%，比活性为2.64 U/mg蛋白。总体纯化倍数约为19,700倍。通过Sephadex G-200凝胶过滤层析发现该酶的分子量为62±3 kDa。在SDS聚丙烯酰胺平板凝胶电泳上获得了一条对应分子量为69.2±3.2 kDa的蛋白带。在聚焦层析中，晶状体葡萄糖-6-磷酸脱氢酶在pI 5.14处出现一个单峰。根据阿伦尼乌斯曲线计算该酶催化反应的活化能为Ea = 5.88 kcal/mol。pH与速度曲线在pH 7.7和9.6处有两个峰。通过双倒数作图和产物抑制研究表明，该酶遵循“有序双双”顺序动力学。通过图形和统计分析，估计KmNADP+、KmG-6-P、KiNADPH、Ki6-PGA分别为0.008±0.002、0.035±0.013、0.173±0.007和1.771±0.160 mM。牛晶状体中葡萄糖-6-磷酸脱氢酶的观察到的动力学行为与其他来源的该酶一致。
