钙蛋白酶A在黑腹果蝇中的表达与活性

Expression and Activity of Calpain A in Drosophila melanogaster.

作者信息

Cardoso Maira, Oliveira Danielle, Araujo Helena

机构信息

Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

出版信息

Methods Mol Biol. 2019;1915:93-101. doi: 10.1007/978-1-4939-8988-1_8.

DOI:10.1007/978-1-4939-8988-1_8

PMID:30617798

Abstract

Detecting calpain activity in Drosophila tissues is a fundamental tool to study calpain function. We use differential centrifugation to prepare membrane- versus cytosol-enriched fractions for measuring calpain activity with the fluorogenic substrate N-LY-AMC. With this method one can measure calpain A activity in wild-type flies and in several mutant fly backgrounds, revealing a strong correlation between in situ membrane distribution and in vitro determined activity measurements. Here we describe the steps for tissue preparation and calpain activity measurement in the Drosophila embryo.

摘要

检测果蝇组织中的钙蛋白酶活性是研究钙蛋白酶功能的一项基本工具。我们使用差速离心法制备富含膜和胞质溶胶的组分，以便用荧光底物N-LY-AMC测量钙蛋白酶活性。通过这种方法，可以测量野生型果蝇和几种突变果蝇背景下的钙蛋白酶A活性，揭示原位膜分布与体外测定的活性测量值之间的强相关性。在这里，我们描述了果蝇胚胎中组织制备和钙蛋白酶活性测量的步骤。