Purification and characterization of assimilatory nitrite reductase from Candida utilis.
Purification and characterization of assimilatory nitrite reductase from Candida utilis.
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产朊假丝酵母同化亚硝酸还原酶的纯化和表征。
DOI：
10.1042/bj3170147
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Gannamani R. Rao
中科院分区：
文献类型：
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作者：
S. Sengupta;M. Shaila;Gannamani R. Rao
许多植物、藻类、酵母和细菌中的硝酸盐同化是由两种酶介导的：硝酸盐还原酶 (EC 1.6.6.2) 和亚硝酸盐还原酶 (EC 1.7.7.1)。它们分别催化硝酸盐逐步还原为亚硝酸盐和亚硝酸盐逐步还原为氨。来自工业上重要的酵母产朊假丝酵母的亚硝酸还原酶已被纯化至均质。纯化的亚硝酸还原酶是异二聚体，两个亚基的分子量分别为 58 和 66 kDa。通过凝胶过滤分析，天然酶的分子量为 126 kDa。使用葫芦叶亚硝酸还原酶抗体通过免疫印迹证实了亚硝酸还原酶的两个亚基的鉴定。通过以下方法证实了编码两个亚基的两个不同大小转录物的存在：(a) 来自硝酸盐诱导的产朊棒杆菌的 mRNA 的体外翻译，随后用异源亚硝酸还原酶抗体对体外翻译产物进行免疫沉淀，以及 (b) Northern 印迹分析。 66 kDa 亚基本质上呈酸性，这可能是由于其磷酸化状态所致。该酶在一定温度范围内稳定。两个亚基都可以催化亚硝酸盐还原，并且重构的酶在较高的蛋白质浓度下表现出与纯化的酶相似的活性。除了大量常见的肽之外，这些亚基中的每一个都已被证明含有一些独特的肽。已发现还原甲基紫精在催化过程中与 NADPH 一样有效的电子供体，这种现象在其他系统的亚硝酸还原酶中并不常见。
Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.