Dupuy Alexander, Passam Freda
Haematology Research Group, Heart Research Institute, Newtown, NSW, Australia.
Cardiovascular Division, Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia.
Methods Mol Biol. 2019;1967:149-163. doi: 10.1007/978-1-4939-9187-7_9.
Endoplasmic reticulum protein 5 (ERp5) is a member of the thiol isomerase family of enzymes, whose prototype member is protein disulphide isomerase (PDI). Thiol isomerases catalyze reduction/oxidation (redox) reactions which lead to the cleavage, formation, or isomerization of disulphide bonds in protein substrates. Thiol isomerase reactions on protein disulphides are important for the correct folding of proteins in the endoplasmic reticulum and for the regulation of various protein functions in the extracellular space. Apart from the disulphide reactions, thiol isomerases assist protein folding by chaperone activity.The disulphide redox activity of ERp5 can be measured with functional assays involving artificial or natural substrates containing disulphide bonds. Herein we describe step-by-step assays of ERp5 reductase, isomerization, and de-nitrosylation activity. Disulphide reductase assays include insulin or di-eosin-GSSG as substrates whereas the isomerization assay includes RNase as substrate. The reduction of natural substrates, i.e., integrin αIIbβ3, can be detected using maleimide labels of free thiols and Western blotting. The biotin switch assay is used to measure the de-nitrosylation of S-nitrosylated substrates. These assays can measure the activity of purified ERp5 protein but can also be applied for the measurement of thiol isomerase activity in cellular samples.
内质网蛋白5（ERp5）是硫醇异构酶家族的成员，该家族的原型成员是蛋白质二硫键异构酶（PDI）。硫醇异构酶催化还原/氧化（氧化还原）反应，这些反应导致蛋白质底物中二硫键的断裂、形成或异构化。蛋白质二硫键上的硫醇异构酶反应对于内质网中蛋白质的正确折叠以及细胞外空间中各种蛋白质功能的调节很重要。除了二硫键反应外，硫醇异构酶还通过伴侣活性协助蛋白质折叠。ERp5的二硫键氧化还原活性可以通过涉及含有二硫键的人工或天然底物的功能测定来测量。本文我们描述了ERp5还原酶、异构化和去亚硝基化活性的逐步测定方法。二硫键还原酶测定以胰岛素或二曙红-GSSG作为底物，而异构化测定以核糖核酸酶作为底物。天然底物即整合素αIIbβ3的还原可以使用游离硫醇的马来酰亚胺标记和蛋白质印迹法进行检测。生物素转换法用于测量S-亚硝基化底物的去亚硝基化。这些测定方法可以测量纯化的ERp5蛋白的活性，但也可用于测量细胞样品中的硫醇异构酶活性。
