用线性动力学方法考察黄嘌呤对尿酸酶的抑制作用  

Inhibitory effects of xanthine on uricase analyzed by a linear kinetic method

作　　者：赵利娜[1] 廖飞[1] 赵运胜[1] 

机构地区：[1]重庆医科大学生物化学教研室,重庆400016

出　　处：《第四军医大学学报》2005年第15期1363-1365,共3页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30472139);重庆医科大学创新基金(2002CX03)

摘　　要：目的:以米氏常数相差较大的两种尿酸酶为模型,考察用两个底物浓度下标定比活性确定酶动力学参数和筛选抑制剂的可靠性.方法:用293nm吸收监测尿酸酶反应.通过两个底物浓度下标定比活性确定表观米氏常数(apparentmichaelismentenconstant,Kmapp)和表观最大反应速度(apparentmaximalreactionrate,Vmapp).据表观动力学参数随抑制剂浓度的变化确定抑制类型和抑制常数(inhibitionconstant,Ki).结果:只要所用尿酸浓度中较小者(thelowerconcentrationofuricacid,SL)大于待测Km的0.8倍且较大者(thehigherconcentrationofuricacid,SH)在SL的1.4倍以上,此线性动力学法能可靠测定Bacillusfastidiosus和Candidaspecies尿酸酶的米氏常数,且与双倒数法结果一致.如SL大于Km的3.8倍而SH为SL的3倍,此线性动力学法所得Candidaspecies尿酸酶Kmapp与抑制剂浓度成正比,Ki为(4.8±0.5)μmol/L,与双倒数法结果一致.Bacillusfastidiosus尿酸酶Km太高,底物浓度有限时不能用此方法筛选其抑制剂.结论:此线性动力学方法仅用高于Km的底物浓度也能可靠确定酶动力学参数,有利于用常规定量方法筛选对底物具有高亲合力特殊靶酶的抑制剂.AIM： To investigate the reliability of a linear kinetic method based on calibrated specific activities of enzyme at two substrate concentrations in estimating apparentk inetic parameters and characterizing inhibitors by using two uricases with different Km as models. METHODS： Uricase reaction was monitored by the absorbance at 293 nm and apparent kinetic parameters （apparent Michaelis-Menten constant, Kmapp, and apparent maximal reaction rate,Vmapp） were estimated based on the calibrated specific activities of uricase at two substrate concentrations. The inhibition type and inhibition constant （K1） were determined by the responses of apparent kinetic parameters to the xanthine concentrations. RESULTS： Km of Bacillus fastidiosus uricase and Candida species uricase were estimated with lower concentration of uric acid （ SL ） above four-fifth of Km and higher concentration of uric acid （ SH ） above 1.4-fold of SL in consistence to double-reciprocal analysis. With SL at 3.8-fold Km and SH at 3-fold SL, there was linear responseof Kmapp of Candida species uricase by this linear kinetic method to xanthine concentrations, and the inhibition constant was （4.8 ± 0.5） μmol/L, consistent to double-reciprocal analysis. This linear kinetic method did not yield reliable inhibition constant of xanthine on Bacillus fastidiosus uricase of much higher Kin. CONCLUSION： This linear kinetic method, reliable in estimating kinetic parameters and characterizing inhibitors of enzyme with lower Km by using two concentrations of the substrate above its Km, makes it possible to use routine technique for quantification.

关 键 词：标定比活性 尿酸酶 黄嘌呤 米氏常数 抑制常数 双倒数分析法 

分 类 号：Q55[生物学—生物化学] Q555.8