In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis
In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis
复制标题
产朊假丝酵母同化亚硝酸还原酶的体外和体内调节
DOI：
10.1007/s002030050491
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发表时间：
1997
影响因子：
2.8
通讯作者：
G. R. Rao
中科院分区：
文献类型：
--
作者：
S. Sengupta;M. Shaila;G. R. Rao
产朊假丝酵母的硝酸盐同化途径与其他同化生物一样，由两种酶介导：硝酸盐还原酶和亚硝酸盐还原酶。纯化的亚硝酸盐还原酶已被证明是由58-和66-kDa亚基组成的异二聚体。在本研究中，亚硝酸盐还原酶被发现能够利用NADH和NADPH作为电子供体。FAD是一种必需的辅酶，在纯化过程中稳定了酶。用半胱氨酸修饰剂对酶进行修饰，巯基试剂可逆转酶的失活。一个半胱氨酸被证明是必不可少的酶活性。在体外，该酶被途径的终产物铵盐灭活，证明该酶具有同化功能。在体内，该酶被硝酸盐诱导，被铵离子抑制。在诱导和阻遏过程中，亚硝酸盐还原酶的mRNA水平、蛋白质水平和酶活性一起受到调节，这表明该酶的主要调节水平是在转录水平。当酶在体外与铵盐一起孵育时，或者当酶在与氮源相同的盐生长的细胞中测定时，残留的酶活性相似。因此，研究产朊假丝酵母亚硝酸还原酶的体外失活，可以为了解产朊假丝酵母亚硝酸还原酶的体内调控机制提供线索。
The nitrate assimilation pathway inCandida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the pathway, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase inCandida utilis.