Warangkar Suchita C, Khobragade Chandrahas N
Biotechnology Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431606, India.
Enzyme Res. 2010;2010:165878. doi: 10.4061/2010/165878. Epub 2009 Nov 1.
L-asparaginase was extracted from Erwinia carotovora and purified by ammonium sulfate fractionation (60-70%), Sephadex G-100, CM cellulose, and DEAE sephadex chromatography. The apparent Mr of enzyme under nondenaturing and denaturing conditions was 150 kDa and 37 ± 0.5 kDa, respectively. L-asparaginase activity was studied in presence of thiols, namely, L-cystine (Cys), L-methionine (Met), N-acetyl cysteine (NAC), and reduced glutathione (GSH). Kinetic parameters in presence of thiols (10-400 μM) showed an increase in V(max) values (2000, 2223, 2380, 2500, and control 1666.7 μmoles mg(-1)min(-1)) and a decrease in K(m) values (0.086, 0.076, 0.062, 0.055 and control 0.098 mM) indicating nonessential mode of activation. K(A) values displayed propensity to bind thiols. A decrease in V(max)/K(m) ratio in concentration plots showed inverse relationship between free thiol groups (NAC and GSH) and bound thiol group (Cys and Met). Enzyme activity was enhanced in presence of thiol protecting reagents like dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and GSH, but inhibited by p-chloromercurybenzoate (PCMB) and iodoacetamide (IA).
L-天冬酰胺酶从胡萝卜软腐欧文氏菌中提取，并通过硫酸铵分级分离（60 - 70%）、葡聚糖凝胶G - 100、CM纤维素和二乙氨基乙基葡聚糖凝胶色谱法进行纯化。在非变性和变性条件下，该酶的表观分子量分别为150 kDa和37±0.5 kDa。在存在硫醇（即L-胱氨酸（Cys）、L-甲硫氨酸（Met）、N-乙酰半胱氨酸（NAC）和还原型谷胱甘肽（GSH））的情况下研究了L-天冬酰胺酶的活性。在存在硫醇（10 - 400 μM）的情况下，动力学参数显示V(max)值增加（分别为2000、2223、2380、2500，对照为1666.7 μmol mg(-1)min(-1)），而K(m)值降低（分别为0.086、0.076、0.062、0.055，对照为0.098 mM），表明为非必需激活模式。K(A)值显示出结合硫醇的倾向。在浓度图中V(max)/K(m)比值的降低表明游离硫醇基团（NAC和GSH）与结合硫醇基团（Cys和Met）之间呈反比关系。在存在二硫苏糖醇（DTT）、2-巯基乙醇（2-ME）和GSH等硫醇保护试剂时，酶活性增强，但受到对氯汞苯甲酸（PCMB）和碘乙酰胺（IA）的抑制。
