摘要
目的获得重组SjLDH主要的酶动力学参数。方法分光光度计测定NADH在340nm处吸光度的变化,检测不同pH及温度下重组蛋白催化正、逆反应的效率以确定其最佳pH、最佳温度;分别固定底物NADH、丙酮酸、NAD+及乳酸浓度,分别测定丙酮酸、NADH、乳酸及NAD+在不同浓度下的反应速度,计算机Lineweaver-Burk双倒数方程回归计算各底物的米氏常数(Km值)、最大反应速度(Vmax),并比较各自的Vmax/Km值。结果重组蛋白酶活性为379U/mg。催化正、逆反应的最佳pH分别为pH6.0～7.0和pH9.0～10.0;催化正、逆反应的最佳温度分别为37～60℃及40～50℃,后者在70℃时仍有较高催化活性;在NADH辅酶作用下,重组SjLDH催化丙酮酸还原为乳酸的最大反应速度是催化NAD+作用下乳酸氧化为丙酮酸的21倍。比较丙酮酸与乳酸的Vmax/Km值,前者是后者的23倍,而NADH的Vmax/Km值是NAD+的7倍。结论重组蛋白在生理条件下主要催化丙酮酸还原为乳酸的反应。
Objective To study the enzyme kinetics of recombinant SjLDH. Method Spectrophotometric method was used to measure the absorbance （340 nm） of NADH. The optimal pH and temperature of recombinant SjLDH to catalyze pyruvate reduction and lactate oxidation reaction were determined. For the analysis of enzyme kinetics, various concentrations of substrates （pyruvate, NADH, lactate, and NAD＋） were used, while the concentrations of the corresponding co-substrates （NADH, pyruvate, NAD＋, lactate） were kept at constant. The results were analyzed by double reciprocal L/neweaver-Burk plot and the kinetic parameters （Km, Vmax, and Vmax/ Km） were calculated from these plots. Result The activity of the recombinant SjLDH was 379 U/mg. The optimal conditions for pyruvate reduction and lactate oxidation reaction was pH 6-7 （37-60℃） and pH 9-10 （40-50℃）, respectively. The maximum rate of pyruvate reduction by NADH was 21-fold faster than the rate of oxidation of lactate by NAD＋. The ratio of Vmax/Km of pyruvate reduction was 23-fold greater than the oxidation of lactate. The ratio of Vmax/Km of NADH-dependent reduction of pyruvate was 7-fold greater than NAD ＋. Conclusion Under the physiological condition, recombinant SjLDH catalyzes the reduction of pyruvate to lactate.
出处
《热带医学杂志》
CAS
2007年第2期122-125,共4页
Journal of Tropical Medicine
关键词
日本血吸虫
乳酸脱氢酶
酶
特性
Schistosome japonicum
lactate dehydrogenase
enzyme
characterization
