豇豆（Vigna unguiculata L. Walp）固氮根瘤中肌苷单磷酸氧化还原酶的纯化及性质

Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp).

作者信息

Atkins C A, Shelp B J, Storer P J

出版信息

Arch Biochem Biophys. 1985 Feb 1;236(2):807-14. doi: 10.1016/0003-9861(85)90687-3.

Abstract

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.

摘要

通过硫酸铵沉淀、凝胶过滤和亲和色谱法，从豇豆（Vigna unguiculata L. Walp）固氮根瘤的植物细胞部分的可溶性蛋白质中分离出肌苷一磷酸（IMP）氧化还原酶（EC 1.2.1.14）。该酶经纯化后比活性提高了140多倍，产率为11%，用甘油稳定，并且需要一种巯基还原剂来达到最大活性。凝胶过滤表明其分子量为200,000，十二烷基硫酸钠-凝胶电泳显示有一个50,000 Da的单一亚基。最终的比活性范围为1.1至1.5 μmol min⁻¹ mg蛋白质⁻¹。该酶的最适pH为碱性，对IMP具有高亲和力（在pH 8.8时Km = 9.1×10⁻⁶ M，NAD水平高于0.25 mM），对NAD也具有高亲和力（在pH 8.8时Km = 18 - 35×10⁻⁶ M）。NAD是首选辅酶，NADP的还原量不到NAD的10%，而分子氧不作为电子受体。脲代谢中间体（尿囊素、尿囊酸、尿酸、肌苷、黄苷和XMP）对该酶无影响，而AMP、GMP和NADH是抑制剂。GMP的抑制作用具有竞争性，Ki = 60×10⁻⁶ M。纯化后的酶被K⁺激活（Km = 1.6×10⁻³ M），但不被NH₄⁺激活。K⁺的激活作用被Mg²⁺竞争性抑制。文中讨论了IMP氧化还原酶的性质对豆科植物根瘤中脲生物合成调节的意义。