水牛肝脏L-精氨酸酶的纯化、表征及其与二氢嘧啶和金属离子的动力学性质

Purification and characterization of buffalo liver L-arginase and its kinetic properties with dihydropyrimidine and metal ions.

作者信息

Nile Shivraj Hariram, Park Se Won

出版信息

Indian J Exp Biol. 2016 Jun;54(6):414-9.

Abstract

Arginase (L-arginine amidinohydrolase, EC.3.5.3.1) from animal tissues such as, liver and kidney has been partially characterized by many researchers. In this study, we purified arginase to homogeneity from buffalo liver with about ~2857 purification fold and a 20% recovery by chromatographic and spectroscopic analysis were obtained. The molecular mass determined by gel filtration and SDS-PAGE was found to be 118 kDa and 47 kDa, respectively. The optimal pH and temperature of the arginase was 9.5 and 40°C, respectively. Kinetic parameters (Km and Vmax) showed activation of arginase in the reaction medium with decrease in Km (7.14, 5.26, 4.0 and control 3.22 mM) and Vmax (0.05, 0.035, 0.027 and control 0.021 mg/mL/min), while co-factor activity of arginase was optimized using metal ions like Mn²⁺ and Mg²⁺ at 2 mM, which revealed an increase in Vmax values (0.011, 0.013, 0.015 and control 0.010 mg/mL/min) and a decrease in Km values (2.22, 2.12, 1.88 and control 1.66 mM). The kinetic data suggested that the arginase activity is enhanced in the presence of dihydropyrimidine derivative and metal ions, indicating essential mode of activation.

摘要

来自肝脏和肾脏等动物组织的精氨酸酶（L-精氨酸脒基水解酶，EC.3.5.3.1）已被许多研究人员部分表征。在本研究中，我们从水牛肝脏中纯化出了纯度达到约2857倍且回收率为20%的均一精氨酸酶，并通过色谱和光谱分析获得了相关结果。通过凝胶过滤和SDS-PAGE测定的分子量分别为118 kDa和47 kDa。精氨酸酶的最佳pH值和温度分别为9.5和40°C。动力学参数（Km和Vmax）显示，在反应介质中精氨酸酶被激活，Km值降低（分别为7.14、5.26、4.0，对照为3.22 mM），Vmax值降低（分别为0.05、0.035、0.027，对照为0.021 mg/mL/min），而精氨酸酶的辅因子活性在2 mM的Mn²⁺和Mg²⁺等金属离子作用下得到优化，这显示Vmax值增加（分别为0.011、0.013、0.015，对照为0.010 mg/mL/min），Km值降低（分别为2.22、2.12、1.88，对照为1.66 mM）。动力学数据表明，在二氢嘧啶衍生物和金属离子存在的情况下，精氨酸酶活性增强，表明其激活的基本模式。