Berrada W, Naya A, Iddar A, Bourhim N
Département de Biologie, Faculté des Sciences, Université Hassan II-Ain Chock, Maârif, Casablanca, Morocco.
Mol Cell Biochem. 2002 Feb;231(1-2):117-27. doi: 10.1023/a:1014464831573.
Cytosolic glycerol-3-phosphate dehydrogenase was purified from jerboa (Jaculus orientalis) skeletal muscle and its physical and kinetic properties investigated. The purification method consisted of a multi-step procedure and this procedure is presented. The specific activity of the purified enzyme is 53.6 U/mg of protein, representing a 77-fold increase in specific activity. The apparent Michaelis constant (Km) for dihydroxyacetone is 137.39 (+/- 25.56) microM whereas the Km for glycerol-3-phosphate is 468.66 (+/- 27.59) microM. The kinetic mechanism of purified enzyme is 'ordered Bi-Bi' and this result is confirmed by the product inhibition pattern. Under the conditions of assay, the pH optimum occurs at pH 7.7 for the reduction of dihydroxyacetone phosphate and at pH 9.0 for glycerol-3-phosphate oxidation. In the direction of dihydroxyacetone phosphate, the optimal temperature is 35 degrees C. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 33,000 (+/- 1000), whereas non-denaturing polyacrylamide gel yields a molecular weight of 72,000 (+/- 2000), suggesting that the enzyme may exist as a dimer. A polyclonal antiserum raised against the purified enzyme was used to localize the enzyme in different jerboa tissues by Western blot method. The purified enzyme is sensitive to N-ethylmaleimide, and incubation of the enzyme with 20 mM N-ethylmaleimide resulted in a complete loss of catalytic activity. The purified enzyme is inhibited by several metal ions including Zn2+ and by 2,4-dichlorophenoxyacetic acid.
从三趾跳鼠（东方三趾跳鼠）骨骼肌中纯化出胞质甘油-3-磷酸脱氢酶，并对其物理和动力学性质进行了研究。纯化方法包括多步骤操作，本文介绍了该操作过程。纯化酶的比活性为53.6 U/mg蛋白质，比活性提高了77倍。二羟基丙酮的表观米氏常数（Km）为137.39（±25.56）μM，而甘油-3-磷酸的Km为468.66（±27.59）μM。纯化酶的动力学机制为“有序双底物双产物”，产物抑制模式证实了这一结果。在测定条件下，磷酸二羟基丙酮还原的最适pH为7.7，甘油-3-磷酸氧化的最适pH为9.0。在磷酸二羟基丙酮方向，最适温度为35℃。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定的纯化酶分子量为33,000（±1000），而非变性聚丙烯酰胺凝胶得到的分子量为72,000（±2000），表明该酶可能以二聚体形式存在。用针对纯化酶制备的多克隆抗血清通过蛋白质印迹法在不同的三趾跳鼠组织中定位该酶。纯化酶对N-乙基马来酰亚胺敏感，用20 mM N-乙基马来酰亚胺孵育该酶会导致催化活性完全丧失。纯化酶受到包括Zn2+在内的几种金属离子以及2,4-二氯苯氧乙酸的抑制。
