Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana)
Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana)
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DOI：
10.1002/arch.10143
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发表时间：
2004-06-01
影响因子：
2.2
通讯作者：
Grossman, SH
中科院分区：
文献类型：
--
作者：
Brown, AE;France, RM;Grossman, SH
本文报道了蟑螂精氨酸激酶的分离与鉴定。纯化方案从6.8 g整只蟑螂中产生6.6 mg纯酶。纯化后的酶与虾精氨酸激酶的异源抗体和单克隆抗体交叉反应。这两种抗体制剂也可以与几种已知含有单体精氨酸激酶的物种的提取物交叉反应，但不能与含有二聚精氨酸激酶的生物体的提取物发生反应。通过凝胶过滤和凝胶电泳测定，蟑螂精氨酸激酶的分子质量约为43,000。与其他精氨酸激酶相比，该酶具有较好的热稳定性（50%的活性在50℃下保持10 min），其最适pH值分别为8.5和6.5-7.5。用5,5'二硫代obis[2-硝基苯甲酸]处理表明精氨酸激酶有一个单一的活性巯基，有趣的是，反应是双相的。磷脂底物（精氨酸：0.49 mM，磷酸甘氨酸：0.94 mM）和核苷酸底物（MgATP: 0.14 mM, MgADP: 0.09 mM）的Michaelis常数在其他精氨酸激酶报道的范围内。1%的纯酶溶液在280 nm处吸光度为7.0。基于圆二向色光谱的计算表明，蟑螂精氨酸激酶具有12%的α -螺旋结构。蛋白质固有荧光发射最大值为340 nm，表明色氨酸残基位于蛋白质表面以下，未暴露于溶剂。从蟑螂和虾中提取的精氨酸激酶是已知的对人体有害的免疫原。纯蛋白质的获得、其特性和潜在的活性调控，将有助于开发控制蟑螂种群及其对农业和人类健康的破坏性作用的药物。(C) 2004 Wiley-Liss, Inc。
The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinarse, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50degreesC for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinarse from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues ore below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health. (C) 2004 Wiley-Liss, Inc.