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Studies on the Specificity and Mechanism of Action of Hepatic Glutathione-Insulin Transhydrogenase

From the ¹ From the Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, and the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014

Studies on the specificity and mechanism of action of hepatic glutathione-insulin transhydrogenase, an enzyme capable of catalyzing the reductive cleavage of the disulfide bonds of insulin in the presence of excess reduced glutathione, have been carried out. By coupling reactions catalyzed by glutathione-insulin transhydrogenase to that of glutathione reductase, it was indicated that during the course of enzyme action, transhydrogenase undergoes reduction by GSH and that the reduced enzyme is autoxidizable; it is inferred that this reduced form is an active intermediate during transhydrogenase-catalyzed reductions of disulfide-substrates. Under conditions of limited spontaneous air oxidation of protein sulfhydryls, transhydrogenase was shown to promote also the net oxidation of such thiols as the reduced forms of insulin and RNase.

During the process of reoxidation of reduced forms of insulin and RNase, glutathione-insulin transhydrogenase was capable of enhancing the rates of regeneration of the native proteins from their reduced precursors as determined by their immunologic and enzymic properties, respectively. Furthermore, this transhydrogenase was able to promote the reconstitution of the native activity from a reduced form of RNase already extensively oxidized by dehydroascorbate. Contrary to previous indications, glutathione-insulin transhydrogenase was found to have no observable activity in specifically directing the preferential re-establishment of the native configuration of insulin.

On the basis of evidence presented in this study, a mechanism involving a series of disulfide-interchange reactions has been proposed to explain the aforementioned reactions catalyzed by glutathione-insulin transhydrogenase. The possible relationship or identity of the transhydrogenase to other insulin-reducing and RNase-reactivating enzymes is pointed out.

In substrate specificity studies on a number of chemically modified and proteolytically degraded forms of insulin, it was found that neither biological activity nor native structure is prerequisite for activity as substrate for glutathione-insulin transhydrogenase-catalyzed reduction.

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