一种基于荧光素酶的5'-腺苷硫酸还原酶检测方法。

A luciferase-based method for assay of 5'-adenylylsulfate reductase.

作者信息

Xiang Xiaoli, Pan Guangtang, Rong Tingzhao, Zheng Zhi-Liang, Leustek Thomas

机构信息

Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA; Institute of Maize Research, Key Laboratory of Biology and Genetic Improvement of Maize in the Southwest Region, Ministry of Agriculture, Sichuan Agricultural University, Chengdu 611130, China.

Institute of Maize Research, Key Laboratory of Biology and Genetic Improvement of Maize in the Southwest Region, Ministry of Agriculture, Sichuan Agricultural University, Chengdu 611130, China.

出版信息

Anal Biochem. 2014 Sep 1;460:22-8. doi: 10.1016/j.ab.2014.05.009. Epub 2014 May 22.

DOI:10.1016/j.ab.2014.05.009

PMID:24857786

Abstract

A luciferase-based method was developed for measurement of 5'-adenylylsulfate (APS) reductase (APR), an enzyme of the reductive sulfate assimilation pathway in prokaryotes and plants. APR catalyzes the two-electron reduction of APS and forms sulfite and adenosine 5'-monophospahate (AMP). The luciferase-based assay measures AMP production using an enzyme-coupled system that generates luminescence. The method is shown to provide an accurate measurement of APR kinetic properties and can be used for both endpoint and continuous assays. APR activity can be measured from pure enzyme preparations as well as from crude protein extracts of tissues. In addition, the assay is ideally suited to high-throughput sample analysis of APR activity in a microtiter dish format. The method adds new capability to the study of the biochemistry and physiology of APR.

摘要

开发了一种基于荧光素酶的方法来测定5'-腺苷硫酸（APS）还原酶（APR），APR是原核生物和植物中还原性硫酸盐同化途径的一种酶。APR催化APS的双电子还原，形成亚硫酸盐和腺苷5'-单磷酸（AMP）。基于荧光素酶的测定使用产生发光的酶偶联系统来测量AMP的产生。该方法被证明能够准确测量APR的动力学特性，可用于终点测定和连续测定。APR活性可以从纯酶制剂以及组织的粗蛋白提取物中进行测量。此外，该测定非常适合以微量滴定板形式对APR活性进行高通量样品分析。该方法为APR的生物化学和生理学研究增添了新的能力。