Oxalate oxidase from barley roots: Purification to homogeneity and study of some molecular, catalytic, and binding properties
Oxalate oxidase from barley roots: Purification to homogeneity and study of some molecular, catalytic, and binding properties
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DOI：
10.1006/abbi.1997.9896
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发表时间：
1997-04-15
影响因子：
3.9
通讯作者：
Clonis, YD
中科院分区：
文献类型：
--
作者：
Kotsira, VP;Clonis, YD
从大麦幼苗根中分离纯化了草酸氧化酶（Oxalate Oxidase，OXO）。纯化方法包括：（i）热处理（60 ℃，10分钟），（ii）在固定的Procion turquoise MX-G染料或仿生氨基乙基草胺蓝染料上的亲和层析，和（iii）在固定的凝集素伴刀豆球蛋白A上的亲和层析（总体性能：1096倍纯化，42%回收率）。纯化的酶具有34 U mg（-1）（25 ℃）的比活性，并且是M-r接近125，000（HPLC分析）的同五聚体，在用银染色后，在SDS-聚丙烯酰胺凝胶电泳上显示单一条带（M-r接近26，000）。纯化的酶对草酸盐的动力学常数为Km 0.27 mM和k（cat）22 s（-1）（37 ℃），而在[草酸盐]大于或等于4 mM时，酶表现出底物抑制。大麦根OXO在370或450 nm处不含辅基吸收，核黄素和FAD对其活性无影响。该酶被Ca 2+（1.7倍）和Pb 2+（2.6倍）各1 mM激活。变性的不可逆失活研究（70 ℃）和天然（37 ℃）酶使用巯基攻击试剂5，5-二硫代双（2-硝基苯甲酸）（1.4 mM），分别在存在和不存在SDS的情况下，已表明变性OXO（4%SDS，10分钟，100 ℃）显示每个分子10个HS基团，而天然OXO在约15分钟孵育后显示每个分子一个可接近的HS基团，并且在相同的时间段内，保持了90%的催化活性。此外，天然OXO处理β-巯基乙醇（1 mM）失去了83%的催化活性在5分钟内。这些研究结果表明，一些半胱氨酸可以保持OXO的催化活性，通过保持其三级结构的完整性，通过二硫键的形成。(C)北京：科学出版社.
Oxalate oxidase (OXO) was purified to homogeneity in three steps from roots of barley seedlings. The purification method comprised: (i) thermal treatment (60 degrees C, 10 min), (ii) affinity chromatography on immobilized either Procion turquoise MX-G dye or biomimetic aminoethyl oxamic blue dye, and (iii) affinity chromatography on immobilized lectin concanavalin A (overall performance: 1096-fold purification, 42% recovery). The purified enzyme has a specific activity of 34 U mg(-1) (25 degrees C), and is a homopentamer of M-r approximate to 125,000 (HPLC analysis) showing a single band on SDS-polyacrylamide gel electrophoresis (M-r approximate to 26,000) after staining with silver nitrate. The kinetic constants of the purified enzyme for oxalate are K-m 0.27 mM and k(cat) 22 s(-1) (37 degrees C), whereas at [oxalate] greater than or equal to 4 mM the enzyme exhibited substrate inhibition. Barley root OXO contains no prosthetic group absorbing at 370 or 450 nm, and riboflavin and FAD have no effect on its activity. The enzyme is activated by 1 mM each of Ca2+ (1.7-fold) and Pb2+ (2.6-fold). Irreversible inactivation studies with denatured (70 degrees C) and native (37 degrees C) enzyme using the sulfhydryl-attacking reagent 5,5-dithiobis(2-nitrobenzoic) acid (1.4 mM), in the presence and absence of SDS, respectively, have shown that denatured OXO (4% SDS, 10 min, 100 degrees C) exhibited 10 HS groups per molecule, whereas native OXO displayed one accessible HS group per molecule after approximately 15 min incubation and, over the same period, maintained its catalytic activity to 90%. Furthermore, native OXO treated with beta-mercaptoethanol (1 mM) lost 83% of its catalytic activity within 5 min. These findings indicate that some cysteines may preserve the catalytic activity of OXO by maintaining the integrity of its tertiary structure via disulfide bond formation. (C) 1997 Academic Press.