Bovine thioredoxin system. Purification of thioredoxin reductase from calf liver and thymus and studies of its function in disulfide reduction.

Abstract

Thioredoxinreductase (EC l&4.5), which catalyzes the reduction of the disulfide bridge in thioredoxin by NADPH, was purified from calf liver and thymus.Preparation methods, involving chromatography on DEAE-cellulose, TEAEcellulose, and Sephadex G-200 or G-100 were used to purify the calf liver enzyme llOO-fold and the thymus enzyme 2800fold.The enzyme was shown to catalyze an NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) which could be used to develop a simple and rapid assay in addition to a specific calf liver thioredoxin-dependent reduction of disulfide bonds in bovine insulin.The purified enzyme, which was inhibited by 0.1 rnM arsenite, showed a wider substrate specificity than the corresponding enzyme from Escherichia coli and rapidly reduced E. coli thioredoxin, yeast thioredoxin, and 5,5'-dithiobis(2nitrobenzoic acid).Phage T4 thioredoxin was slowly reduced.The apparent k,,, values for 5,5'-dithiobis(2-nitrobenzoic acid) and calf liver thioredoxin were 1.5 mM and 5 FM, respectively.In vitro oxidized preparations of calf liver thioredoxin were shown to contain high molecular weight mixed disulfide aggregates that were reduced by the enzyme with kinetics which supported a process of autoactivation.This may be of importance as a control mechanism for the activity of the bovine thioredoxin system.Reduction of disulfide bonds in insulin or L-cystine by NADPH and thioredoxin reductase was absolutely dependent upon the presence of thioredoxin as intermediate electron carrier.With the coupled system, fast reduction of