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

中国临床药理学与治疗学 ›› 2008, Vol. 13 ›› Issue (10): 1127-1133.

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

周永列¹, 吕亚萍², 许武林¹, 胡惟孝²

¹浙江省人民医院中心实验室;
²浙江工业大学药学院, 杭州 310014, 浙江

收稿日期:2008-05-28 修回日期:2008-07-29 出版日期:2008-10-26 发布日期:2020-10-19
作者简介:周永列, 男, 副教授, 硕士生导师, 研究方向:实验血液学。Tel:0571-85893266 　 E-mail:lab_zyl@126.com
基金资助:
浙江省医学重点学科建设资助项目(07-010); 浙江省医药卫生资助项目(2007A008)

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

ZHOU Yong-lie¹, LV Ya-ping², XU Wu-lin¹, HU Wei-xiao²

¹Clinical Laboratory Medicine Center of Zhejiang Provincial People's Hospital;
²College of Pharmacy of Zhejiang In-dustry University, Hangzhou 310014, Zhejiang, China

Received:2008-05-28 Revised:2008-07-29 Online:2008-10-26 Published:2020-10-19

摘要/Abstract

摘要： 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;0.3 g/L SDS仅对 CT-L 活性有激活作用;CT-L 、T-L 、PGPH-L 的K m 分别为 3.55、4.12、4.88 μmol/L;酶反应进程曲线的线性期达 20 min;线性范围为测定纯 20S蛋白酶体时, 其蛋白浓度分别达 100、120、100 μg/L, 测定 B16 细胞蛋白酶体提取物时, 其蛋白浓度均达 200 mg/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 IC 50 分别为 12.36、 12.42、24.40 nmol/L ;测定 ZGDHu-1 对 20S 蛋白酶体抑制活性, IC 50 分别为 2.24、0.92、0.51 μmol/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。

关键词: 连续荧光监测法, 蛋白酶体, 抑制剂, 硼替佐米, 四嗪二甲酰胺

Abstract: 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 (PG-PH-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 and the technolo-gies were evaluated .The inhibition of the CT-L, T-L and PGPH-L activities of purified 20S proteasome ac-tivity of proteasome extracts were prepared from B16 and Namalwa cells by PS-341 and ZGDHu-1were mea-sured. RESULTS: The optimum pH of enzyme reac-tion was 8.2.The addition of 0.3 g/L SDS buffer only to the assay was required to activate the CT-L of pro-teasome .The K m of CT-L, T-L and PGPH-L activities of proteasome were 3.55, 4.12, 4.88 μmol/L respec-tively and the linearity of reaction curve was up to 20 min.When purified 20S proteasome was used, the as-say for detecting CT-L, T-L and PGPH-L activities was linearly dependent on total protein 100, 120, 100 μg/L in 2 mL assay volume respectively .When the extracts of B16 cell proteasome were detected, the proteinum concentrations were 200 mg/L .The degree of precision in within-run and between-run CVs were (2.25-5.78) %and (3.75-8.42) %respectively .The IC 50 values of the PS -341 and ZGDHu-1 for inhibtion of the CT-L, C-L and PGPH-L activities of purified 20S proteasome were 12.36, 12.42, 24.40 nmol/L and 2.24, 0.92, 0.51 μmol/L respectively.CONCLUSION: The assay provides a reliable and effective method for studying the screen of proteasome inhibitors and it is an useful index in clinical trials with protea-some inhibitors.

Key words: fluorescence kinetic assays, protea-some, inhibitor, Bortezomib, N,N'-di-(m-methylphe-nyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide

中图分类号:

R965.2

周永列, 吕亚萍, 许武林, 胡惟孝. 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[J]. 中国临床药理学与治疗学, 2008, 13(10): 1127-1133.

ZHOU Yong-lie, LV Ya-ping, XU Wu-lin, HU Wei-xiao. Determination of proteasome activities with fluorescence kinetic assay and its application in screening proteasome inhibitors[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13(10): 1127-1133.

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