中文摘要：

采用Sephadex G-100凝胶过滤、DEAE Sepharose离子交换、Sephadex G-25凝胶过滤等方法对黄颡鱼肝脏胞质中NADP依赖性的异柠檬酸脱氢酶（IDPc）进行纯化,并研究其相关的酶学性质。结果显示,IDPc的比活力为7.94 U/mg,亚基分子量为36.7 ku。IDPc活性最大时的pH和温度分别为8.0和65℃,活化能为81.33 kJ/mol,底物米氏常数KmNADP和KmIC分别为0.056和0.175 mmol/L,底物最大反应速率VmNADP和VmIC分别为9.04和10.51U/mg,催化效率KcatNADP和KcatIC分别为0.16和0.06 min/mg,产物NADPH对IDPc表现为竞争性抑制,抑制常数KiNADPH为0.034 mmol/L。IDPc的催化作用强烈依赖于金属离子Mn^2＋、Mg^2＋,没有金属离子存在时反应几乎不进行,二价金属离子激活IDPc的顺序为Mn^2＋〉Mg^2＋〉Zn^2＋〉Ca^2＋〉Cu^2＋,Cu^2＋几乎不能激活IDPc的活性。Ca^2＋和Zn^2＋既是激活剂也是抑制剂,与其作用浓度有关,Cu^2＋基本上对其无影响。通过对IDPc系统的酶学性质探讨,能够为深入研究IDPc催化与调节机制奠定基础。

英文摘要：

Nicotinamide-Adenine Dinucleotide Phosphate-Dependent Isocitrate Dehydrogenase （IDPc）is an important enzyme essential for survival of all organisms. Many studies have been conducted to isolate IDPc and explore its kinetic parameters in mammals, plants and microorganisms. However, in fish, the related information is very scarce. The aim of the study is to purify and characterize IDPc from yellow catfish hepatic cytoplasm, which will provide some crucial information on the catalytic and regulatory mechanism of the enzyme in liver of yellow catfish. The purification processes include permeation chromatography on Sephadex G-100 gel, chromatographic adsorption by DEAE Sepharose, permeation chromatography on Sephadex G-25 gel and finally chromatographic adsorption by DEAE Sepharose again. The specific IDPc activity is 7.94 U/mg, and its molecular weight after SDS polyacrylamide slab gel electrophoresis is 36.7 ku. The optimum pH, and temperature for the enzyme are 8.0 and 65 ~（7 respectively. Using Arrhenius plots, the enzyme has the activation energy of 81.33 kJ/mol. The Km values of the substrates NADP＋ and IC are 0. 056 mmol/L and 0. 175 mmol/L, respectively. The Vmax values of IDPc with NADP＋ and IC as the substrates are 9.04 U/mg and 10.51 U/mg,respectively. The enzyme of IDPc is inhibited by NADPH in a competitive manner with the K, value of 0. 034 mmol/L. The activity of IDPc is greatly dependent on the binding of divalent metal ions with the active order of Mn^2 ＋ 〉 Mg^2 ＋ 〉 Zn^2 ＋ 〉 Ca^2 ＋ 〉 Cu^2 ＋ . Ca^2 ＋ and Zn^2 ＋ are both the activator and inhibitor of IDPc, however, Cu2＋ showed no effects on the enzyme. The comprehensive information of enzymatic properties may help to better understand the mechanism of catalysis and regulation of IDPc in fish.