THE CONVERSION OF NITRITE TO NITROGEN OXIDE(S) BY THE CONSTITUTIVE NAD(P)H-NITRATE REDUCTASE ENZYME FROM SOYBEAN
THE CONVERSION OF NITRITE TO NITROGEN OXIDE(S) BY THE CONSTITUTIVE NAD(P)H-NITRATE REDUCTASE ENZYME FROM SOYBEAN
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DOI：
10.1104/pp.88.2.389
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发表时间：
1988-10-01
期刊：
影响因子：
7.4
通讯作者：
HARPER, JE
中科院分区：
文献类型：
--
作者：
DEAN, JV;HARPER, JE
采用两步纯化方法，从大豆(Glyine max[L.])提取的一氧化氮和二氧化氮(NO(X))放出活性中分离出NAD(P)H-硝酸还原酶[NAD(P)H-NR，pH 6.5；EC 1.6.6.2]。(Merr.)传单。无论营养生长条件如何，这两种活性都可以用NADPH从装载野生型或LNR-5和LNR-6(缺乏结构性NADH-NR[pH 6.5])突变大豆植株提取物的蓝色琼脂糖柱上洗脱。蓝色琼脂糖亲和层析后的快速蛋白质液相-阴离子交换(Mono Q柱)层析也未能将两种活性分离。这些数据提供了强有力的证据，证明大豆中的结构性NAD(P)H-NR(pH 6.5)是负责NO(X)形成的酶。该酶的最适pH为6.75，对亚硝酸盐的表观Km为0.49毫摩尔，对NADPH和NADH的表观Km分别为7.2和7.4微摩尔。除NAD(P)H外，还原的黄素单核苷酸(FMNH2)和还原的甲基紫精(MV)还可以作为NO(X)释放活性的电子供体。氰化物均能抑制NADPH-、FMNH2-和MV-NO(X)的释放活性。对羟基苯甲酸汞对NADPH活性也有抑制作用，而FMNH2和MV活性对抑制作用相对不敏感。这些数据表明，酶的末端含钼部分参与了将亚硝酸盐还原为NO(X)的过程，NADPH从负载硝酸盐或零N四季豆提取物的Blue Sepharose柱上洗脱了NR和NO(X)的放出活性，而NADH没有洗脱这两种活性。四季豆似乎只含有一种与大豆NAD(P)H-NR(pH 6.4)组成酶相似的NR酶。
A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO(x)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO(x) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent Km for nitrite of 0.49 millimolar, and an apparent Km for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO(x) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH2) and reduced methyl viologen (MV) can serve as electron donors for NO(x) evolution activity. The NADPH-, FMNH2-, and reduced MV-NO(x) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymercuribenzoate, whereas, the FMNH2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO(x), NADPH eluted both NR and NO(x) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.4) enzyme of soybean.