Thiol protein disulphide oxidoreductases: assay of microsomal membrane bound glutathione insulin transhydrogenase and comparison with protein disulphide isomerase

Abstract

Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane bound glutathione insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. Membrane bound glutathione insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide isomerase (EC 5.3.4.1) is drastically inhibited. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione insulin transhydrogenase and protein disulphide isomerase activities are not both catalysed by a single enzyme species.