连续荧光监测法测定蛋白酶体活性及其在筛选蛋白酶体抑制剂中的应用

Determination of proteasome activities with fluorescence kinetic assay and its application in screening proteasome inhibitors

目的:建立用连续荧光监测法测定蛋白酶体活性的方法,研究其在筛选蛋白酶体抑制剂中的应用价值。方法:用特异性荧光多肽底物Suc-Leu-Leu-Val-Tyr-AMC(Suc-LLVT-AMC)、Z-Val-Val-Arg-AMC(ZVVA-AMC)、Z-Leu-Leu-Glu-βNA(ZLLG-βNA),分别测定蛋白酶体的糜凝乳蛋白酶样(chy-motrypsin-like,CT-L)、胰蛋白酶样(trypsin-like,T-L)和肽基谷氨酰肽水解酶样(PGPH-like,PGPH-L)活性,对测定条件优化,并进行方法学评价。用该法测定了硼替佐米(PS-341)和四嗪二甲酰胺(ZG-DHu-1)对纯20S蛋白酶体和B16、Namalwa细胞蛋白酶体提取物抑制作用。结果:连续荧光监测法测定蛋白酶体活性的最适pH为8.2;0.3g/LSDS仅对CT-L活性有激活作用;CT-L、T-L、PGPH-L的Km分别为3.55、4.12、4.88μmol/L;酶反应进程曲线的线性期达20min;线性范围为测定纯20S蛋白酶体时,其蛋白浓度分别达100、120、100μg/L,测定B16细胞蛋白酶体提取物时,其蛋白浓度均达200mg/L;精密度的批内CV为(2.25～5.78),批间CV在(3.75～8.42)。用该法测定了PS-341对20S蛋白酶体CT-L、T-L、PGPH-L的抑制活性,其IC50分别为12.36、12.42、24.40nmol/L;测定ZGDHu-1对20S蛋白酶体抑制活性,IC50分别为2.24、0.92、0.51μmol/L。结论:连续荧光监测法测定蛋白酶体活性为快速、有效、大规模筛选蛋白体抑制剂类药物提供了一个技术平台,也为临床上应用蛋白酶体抑制剂治疗提供了一个有用的检测指标。

AIM:To establish a method for the determination of proteasome activities with fluorescence kinetic assay and evaluate the application in screening proteasome inhibitors.METHODS:The proteasome activities of chymotrypsin-like(CT-L),trypsin-like(T-L) and peptidyl glutamyl peptide hydrolysing-like(PGPH-L) were measured with specificity fluorescence peptide substrate Suc-Leu-Leu-Val-Tyr-AMC(Suc-LLVT-AMC),Z-Val-Val-Arg-AMC(ZVVA-AMC),Z-Leu-Leu-Glu-βNA(ZLLG-βNA).The conditions of enzyme reaction had been optimised ...