豌豆种子(Pisum sativum)萌发过程中与 NADP+ 链接的异柠檬酸脱氢酶的分离和特征描述。

IF 1.5 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
P K Srivastava, D S Singh
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引用次数: 0

摘要

从发芽的豌豆种子中纯化出了 NADP+ 链接的异柠檬酸脱氢酶(E.C.1.1.1.42)。该酶是一种四聚体蛋白(摩尔质量约为 146,000 ),由明显相同的单体(亚单位摩尔质量约为 36,000 )组成。纯化酶在 45 摄氏度和 50 摄氏度下的热失活显示出简单的一阶动力学。底物 [S] 在不同 pH 值(6.5-8)下对酶活性的影响表明,质子在形式上表现为 "非竞争性抑制剂"。在这个 pH 值范围内,只有底物存在时,酶的一个碱性基团(位点)才会质子化,pKa 等于 6.78。在 0 摄氏度下与乙二胺四乙酸(EDTA)和 pH 值为 7.8 的磷酸盐缓冲液进行连续透析,会产生一种无酶活性的蛋白质,其热失活动力学与未处理的(原生)酶相同。在 Mn2+ 和 Mg2+ 离子(3.75 毫摩尔)存在时,酶活性最大。加入 Zn2+、Cd2+、Co2+ 和 Ca2+ 离子可使酶活性部分恢复。其他金属离子 Fe2+、Cu2+ 和 Ni2+不起作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum).

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.

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来源期刊
Indian journal of biochemistry & biophysics
Indian journal of biochemistry & biophysics 生物-生化与分子生物学
CiteScore
2.90
自引率
50.00%
发文量
88
审稿时长
3 months
期刊介绍: Started in 1964, this journal publishes original research articles in the following areas: structure-function relationships of biomolecules; biomolecular recognition, protein-protein and protein-DNA interactions; gene-cloning, genetic engineering, genome analysis, gene targeting, gene expression, vectors, gene therapy; drug targeting, drug design; molecular basis of genetic diseases; conformational studies, computer simulation, novel DNA structures and their biological implications, protein folding; enzymes structure, catalytic mechanisms, regulation; membrane biochemistry, transport, ion channels, signal transduction, cell-cell communication, glycobiology; receptors, antigen-antibody binding, neurochemistry, ageing, apoptosis, cell cycle control; hormones, growth factors; oncogenes, host-virus interactions, viral assembly and structure; intermediary metabolism, molecular basis of disease processes, vitamins, coenzymes, carrier proteins, toxicology; plant and microbial biochemistry; surface forces, micelles and microemulsions, colloids, electrical phenomena, etc. in biological systems. Solicited peer reviewed articles on contemporary Themes and Methods in Biochemistry and Biophysics form an important feature of IJBB. Review articles on a current topic in the above fields are also considered. They must dwell more on research work done during the last couple of years in the field and authors should integrate their own work with that of others with acumen and authenticity, mere compilation of references by a third party is discouraged. While IJBB strongly promotes innovative novel research works for publication as full length papers, it also considers research data emanating from limited objectives, and extension of ongoing experimental works as ‘Notes’. IJBB follows “Double Blind Review process” where author names, affiliations and other correspondence details are removed to ensure fare evaluation. At the same time, reviewer names are not disclosed to authors.
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