用线性动力学方法表征L-苯丙氨酸对兔小肠黏膜碱性磷酸酶的抑制作用

Reliability of a linear kinetic method in characterizing the inhibition of L-phenylalanine on rabbit intestinal alkaline phosphatase

目的:考察用线性动力学方法表征L-苯丙氨酸对兔小肠黏膜碱性磷酸酶抑制作用的可靠性.方法:测定碱性磷酸酶水解对硝基苯酚磷酸酯(PNPP)的产物生成初速度.用两个底物浓度下标定比活性计算米氏常数(Km)和最大反应速度(Vm),再据二者对抑制剂浓度的响应确定抑制剂类型和抑制常数.结果:双倒数分析法重复测定发现70%的Km和Vm随抑制剂浓度升高而同步下降,两动力学参数变化对应抑制常数的比值为(2.4±0.6,n=9).线性动力学方法所得参数精度稍高,Km和Vm随抑制剂浓度升高同时下降的出现率约80%,两抑制常数之比为(2.0±0.8,n=10),两参数变化对应的抑制常数及两抑制常数之比都与双倒数分析法一致.如允许两抑制常数之比在2.5倍内波动,则两种方法都表明L-苯丙氨酸为此碱性磷酸酶的反竞争性抑制剂.结论:此线性动力学方法可用于表征反竞争性抑制剂.

AIM： To investigate the reliability of a linear kinetic method based on calibrated specific activities of enzyme at two medium substrate concentrations in characterizing the inhibition of L-phenylalanine on rabbit intestinal alkaline phosphatase. METHODS： Phosphatase reaction was monitored by absorbance at 410 nm using p-Nitrophenyl phosphate （PNPP） as substrate. Kinetic parameters （Michaelis-Menten constant, Km, and maximal reaction rate, Vm ） were estimated by this linear kinetic method at two substrate concentrations above KIn. Inhibition type and inhibition constants （ Kik for the change of Km and Kiv for the change of Vm, respectively） were determined by responses of kinetic parameters to phenylalanine concentration. RESULTS： By double-reciprocal analysis, about 70% of Km and Vm decreased simultaneously in response to the increase of L-phenylalanine concentration and Kiv/Kik was （2.4 ±0.6, n =9）. By this linear kinetic method, nearly 80% of kinetic parameters showed similar changes aside from their improved precision. There was no difference in either inhibition constant between this linear kinetic method and double-reciprocal analysis. This linear kinetic method yielded Kiv/Kik of （2.0 ±0. 8, n = 10）, consistent to that by double-reciprocal analysis. Given variation in Kiv/Kik 〈 2.5 were acceptable for uncompetitive inhibition, both methods consistently suggested that L-phenylalanine was an uncompetitive inhibitor on rabbit intestinal alkaline phosphatase. CONCLUSION： This linear kinetic method is applicable to characterize uncompetitive inhibitors.