PURIFICATION AND CHARACTERIZATION OF THE ASSIMILATORY NITRATE REDUCTASE OF AZOTOBACTER-VINELANDII
PURIFICATION AND CHARACTERIZATION OF THE ASSIMILATORY NITRATE REDUCTASE OF AZOTOBACTER-VINELANDII
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DOI：
10.1042/bj2890335
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发表时间：
1993-01-15
影响因子：
4.1
通讯作者：
EADY, RR
中科院分区：
文献类型：
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作者：
GANGESWARAN, R;LOWE, DJ;EADY, RR
1.通过硝酸还原酶活性染色和非变性条件下聚丙烯酰胺凝胶上考马斯亮蓝染色蛋白条带的重合，从硝酸盐上好氧生长的棕色固氮菌UW136菌株中纯化出一种可溶的还原甲基紫精同化硝酸还原酶。比活力为每毫克蛋白质产生3摩尔亚硝酸根/分钟。2.Superose-12凝胶过滤和SDS/PAGE分析表明，该酶的M(R)为105000，为单体。该酶每个分子含有1个Mo原子、4个Fe原子和4个不耐酸的硫化物原子；没有发现细胞色素或FAD存在的证据。3.钼存在于一种钼辅助因子中，它在提取时能够激活粗面脉孢菌nit-1突变体粗提液中发出的apo-(nit-1)硝酸还原酶。4.经分离，该酶具有EP.R.分配给Mo(V)的信号g1=2.023；g2=1,998；g3=1.993，g(av.)=2.004，表明该酶中钼的异常环境。5.用S2O42还原-漂白E.P.R.在加入NO_3~(2-)引发酶周转时，信号表现出与异化硝酸还原酶相似的短时间Mo(V)信号，g1=1.998；g2=1.989；g3=1.981，g(Av)=1.989。随后，延长孵化时间得到了两种EPR的混合物。物种。6.NADH和NADPH都不是有效的电子供体，但还原甲基紫精(表观K(M)988um)和还原溴酚蓝(表观K(M)158um)是有效的。这些供体对硝酸盐的表观K(M)值分别为70um和217um。
1. A soluble reduced Methyl Viologen-dependent assimilatory nitrate reductase from Azotobacter vinelandii strain UW136 grown aerobically on nitrate was purified to homogeneity by the criteria of nitrate reductase activity staining, and coincidence of a Coomassie Blue-staining protein band on polyacrylamide gels run under non-denaturing conditions. The specific activity was 3 mumol of NO2- formed/min per mg of protein. 2. Gel filtration on Superose-12 and SDS/PAGE showed that the enzyme had an M(r) of 105000 and was monomeric. The enzyme contained 1 Mo atom, 4 Fe atoms and 4 acid-labile sulphide atoms per molecule; no evidence for the presence of cytochrome or FAD was found. 3. Mo was present in a molybdenum cofactor, which on extraction was capable of activating apo-(nit-1) nitrate reductase sent in crude extracts of nit-1 mutants of Neurospora crassa. 4. As isolated, the enzyme had e.p.r. signals assigned to Mo(V) with g-values g1 = 2.023; g2 = 1,998; g3 = 1.993 and with g(av.) = 2.004 indicating an unusual environment of Mo in this enzyme. 5. Reduction with S2O42- bleached the e.p.r. signals which, on reoxidation after the addition of NO32- to initiate enzyme turnover, exhibited at short times Mo(V) signals similar to those of dissimilatory nitrate reductases, with g1 = 1.998; g2 = 1.989; g3 = 1.981 and g(av) = 1.989. Prolonged incubation subsequently gave a mixture of both e.p.r. species. 6. Neither NADH nor NADPH was effective as an electron donor, but reduced Methyl Viologen (apparent K(m) 988 muM) and reduced Bromophenol Blue (apparent K(m) 158 muM) were effective. With these donors the apparent K(m) values for nitrate were 70 muM and 217 muM respectively.