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

Thioredoxin reductase (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 insulin was obtained with as little as 3 x 10mH .M calf liver thioredoxin. By a number of criteria the bovine thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) was identical with the enzyme NADPH-protein disulfide reductase and is suggested to play a major role in metabolic oxidoreductions of protein disulfides.