In vitro fluorescent substrate assay for the activity of leucine aminopeptidase(LAP)in Echinococcus multilocularis
10.3969/j.issn.1002-2694.2025.00.010
- VernacularTitle:一种多房棘球绦虫亮氨酸氨基肽酶体外荧光底物酶活性检测方法
- Author:
Jia-yu CHEN
1
;
Yao DAI
;
Shun-juan WANG
;
Yang XIAO
;
Xin-zong YAN
;
Tong LIU
;
Zhi-hao YUAN
;
Kai-li SHI
;
Run-le LI
;
Feng TANG
Author Information
1. 青海大学高原医学研究中心,青海省高原医学应用基础重点实验室,西宁 810001;青海大学研究生院,西宁 810001
- Publication Type:Journal Article
- Keywords:
Echinococcus multilocularis;
LAP;
Leu-AMC;
enzyme activity
- From:
Chinese Journal of Zoonoses
2025;41(1):23-31
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.
