Streit L, Nelson R S, Harper J E
Department of Agronomy, University of Illinois, Urbana, Illinois 61801.
Plant Physiol. 1985 May;78(1):80-4. doi: 10.1104/pp.78.1.80.
nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr(1)-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c(1)NR, and the form eluted with NADH was designated c(2)NR. Nitrate-grown nr(1) mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c(1)NR and c(2)NR had similar pH optima of 6.5, sedimentation behavior (s(20,w) of 5.5-6.0), and electrophoretic mobility. However, c(1)NR was more active with NADPH than with NADH, while c(2)NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c(1)NR) and 0.19 millimolar (c(2)NR). The iNR from the mutant had a pH optimum of 7.5, s(20,w) of 7.6, and was less mobile on polyacrylamide gels than c(1)NR and c(2)NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.
从野生型大豆（Glycine max [L.] Merr.，品种Williams）和nr(1) - 突变型大豆植株中纯化出了NADH：硝酸还原酶（EC 1.6.6.1）和NAD(P)H：硝酸还原酶（EC 1.6.6.2）。纯化过程包括使用完全展开的单叶丙酮粉作为酶源，通过蓝琼脂糖柱和羟基磷灰石柱色谱法。从以尿素作为唯一氮源生长的野生型植物提取物加载的蓝琼脂糖柱中，两种组成型硝酸还原酶形式依次用NADPH和NADH洗脱。用NADPH洗脱的形式被指定为c(1)NR，用NADH洗脱的形式被指定为c(2)NR。当用NADH洗脱蓝琼脂糖柱时，硝酸盐培养的nr(1)突变型大豆植株产生了一种NADH：硝酸还原酶（指定为iNR）；NADPH未能从加载该提取物的蓝琼脂糖柱上洗脱任何NR形式。c(1)NR和c(2)NR都具有相似的最适pH值6.5、沉降行为（s(20,w)为5.5 - 6.0）和电泳迁移率。然而，c(1)NR对NADPH的活性比对NADH更高，而c(2)NR更倾向于以NADH作为电子供体。硝酸盐的表观米氏常数分别为5毫摩尔（c(1)NR）和0.19毫摩尔（c(2)NR）。突变体的iNR最适pH值为7.5，s(20,w)为7.6，在聚丙烯酰胺凝胶上的迁移率比c(1)NR和c(2)NR低。iNR相对于NADPH更倾向于NADH，其硝酸盐的表观米氏常数为0.13毫摩尔。因此，野生型大豆含有两种组成型硝酸还原酶形式，它们的物理性质都与高等植物中常见的硝酸还原酶不同。然而，大豆中存在的诱导型硝酸还原酶形式似乎与高等植物中发现的大多数底物诱导型硝酸还原酶相似。
