定量检测果蝇谷氨酸合成酶 1 的活性。

Quantitation of Glutamine Synthetase 1 Activity in Drosophila melanogaster.

机构信息

Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy.

Department of Anatomy and Cell Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA.

出版信息

Methods Mol Biol. 2023;2675:237-260. doi: 10.1007/978-1-0716-3247-5_18.

DOI:10.1007/978-1-0716-3247-5_18

PMID:37258768

Abstract

Protocols to assay the activity of glutamine synthetase (GS) are presented as they have been used in our laboratory to correlate the expression levels of the gene encoding Drosophila GS1 gene, the GS1 protein levels, and its activity in extracts of larvae and heads from Drosophila melanogaster. The assays are based on the glutamine synthetase-catalyzed formation of γ-glutamylhydroxylamine in the presence of ATP, L-glutamate, and hydroxylamine, in which hydroxylamine substitutes for ammonia in the reaction. Formation of γ-glutamylhydroxylamine is monitored spectrophotometrically in discontinuous assays upon complex formation with FeCl. Fixed-time assays and those based on monitoring the time-course of product formation at different reaction times are described. The protocols can be adapted to quantify glutamine synthetase activity on biological materials other than Drosophila.

摘要

检测谷氨酰胺合成酶（GS）活性的方法如下，这些方法已在我们的实验室中使用，以关联编码果蝇 GS1 基因的基因表达水平、GS1 蛋白水平及其在黑腹果蝇幼虫和头部提取物中的活性。这些测定基于谷氨酰胺合成酶催化在 ATP、L-谷氨酸和羟胺存在下形成γ-谷氨酰羟胺，其中羟胺在反应中替代氨。在不连续测定中，通过与 FeCl 形成复合物来监测γ-谷氨酰羟胺的形成，并通过分光光度法进行监测。描述了固定时间测定法和基于在不同反应时间监测产物形成时间过程的测定法。这些方案可以适应于除果蝇以外的生物材料来定量谷氨酰胺合成酶活性。