Dabir Snehal, Dabir Pankaj, Somvanshi Baburao
Department of Biochemistry, Dr BA Marathwada University, Aurangabad-400001 (MS), India.
Int J Biol Sci. 2005;1(3):114-22. doi: 10.7150/ijbs.1.114. Epub 2005 Aug 1.
Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn(++) ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.
通过使用Bio-Gel P-150、DEAE-纤维素和精氨酸AH Sepharose 4B亲和柱进行色谱分离，从豇豆种子叶和水牛肉肝中纯化出精氨酸酶。在Bio-Gel P-300柱上估计的豇豆种子叶酶的天然分子量为210 kDa，水牛肉肝的为120 kDa，而SDS-PAGE显示种子叶精氨酸酶的单一条带分子量为52 kDa，水牛肉肝精氨酸酶的为43 kDa。对纯化的种子叶和肝脏精氨酸酶测定的动力学性质表明，其最适pH分别为10.0和9.2。发现种子叶精氨酸酶的最佳辅因子Mn(++)离子浓度为0.6 mM，肝脏精氨酸酶的为2 mM。豇豆种子叶精氨酸酶和肝脏精氨酸酶的米氏常数分别为42 mM和2 mM。胍基化合物作为豇豆种子叶和水牛肉肝精氨酸酶的替代底物的活性严重依赖于氨基酸侧链的长度和碳原子数。除L-精氨酸外，种子叶精氨酸酶对胍丁胺和L-刀豆氨酸表现出底物特异性，而肝脏精氨酸酶仅对L-刀豆氨酸表现出底物特异性。
