Protein Disulfide Oxidoreductase Research Articles

Insulin degradation: II. The widespread distribution of glutathione-insulin transhydrogenase in the tissues of the rat

Glutathione-insulin transhydrogenase (glutathione: protein-disulfide oxidoreductase, EC 1.8.4.2) has previously been isolated from liver, kidney and pancreas. In view of the known ability of a variety of other tissues to degrade insulin in vitro, the present systematic survey of rat tissues for the presence of glutathione-insulin transhydrogenase was undertaken. Tissue extracts prepared in potassium phosphate-EDTA buffer were tested for their ability to make soluble the radioactivity of [ 125I]insulin in 5% trichloroacetic acid. In the presence of I mM glutathione, insulin-degrading activity was detected in all tissues studied; the relative activities (per mg of tissue protein) were found to be in the order; pancreas > liver > intestine > spleen > kidney, testis, thymus, fat, lung > brain > heart > diaphragm, skeletal muscle. With each tissue extract, insulin degradation was completely blocked by the addition of N-ethyl-maleimide. Apparent K m values for insulin and for glutathione were similar in all of the tissue extracts. The presence of glutathione-insulin transhydrogenase was demonstrated in all tissue extracts by double immuno-diffusion with antibody to purified glutathione-insulin transhydrogenase. The data indicate that most or all of the insulin degrading activity observed under the experimental conditions used is due to the presence of glutathione-insulin transhydrogenase. The results indicate the possibility that glutathione-insulin transhydrogenase may be the major insulin-degrading activity present in the animal.

Thiol-protein disulfide oxidoreductase activity in human placental tissue homogenates.

Thiol–protein disulfide oxidoreductase activity was detected in the soluble cell fraction of human placental tissue homogenized in sucrose. This activity was demonstrated in the rapid reduction of oxytocin and the somewhat less rapid reduction of insulin by reduced glutathione. The apparent pH optimum of the enzymic activity for the reduction of oxytocin and insulin was found to be near pH 8.

Degradation of oxytocin by human placental tissue

Incubation studies using placental homogenates and oxytocin or its analogue, deamino-oxytocin, show that the “plasma oxytocinase” present in the tissue does not contribute appreciably to the observed degradation of the hormone. This is ascribed to the action of the “tissue oxytocinase” system, made up of a thiol: protein-disulfide oxidoreductase and an aminopeptidase component. The relative significance of the two different oxytocinases in the metabolism of oxytocin in the intact placenta cannot be assigned from these studies.

Acceleration of regeneration of insulin activity from its inactive reduced A and B chains by pancreatic glutathioneinsulin transhydrogenase

1. 1.|Data are presented which show that GSH-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase, EC 1.8.4.2) which promotes the cleavage of disulfide bonds of insulin is also capable of promoting the regeneration of insulin activity from inactive reduced A and B chains of insulin by catalyzing sulfhydryl-disulfide interchange. With the conditions used, insulin activity regained was about 15 times as much in the presence as in the absence of transhydrogenase. There was complete agreement in the estimates of regenerated insulin activity between immunoassay and fat pad assay. 2. 2.|When the reduced chains were allowed to oxidase individually, it was observed the rate of disappearance of 2 of the 4 SH groups of A chain was faster than that of the other 2. In the case of B chain, there was a rapid loss of 0.3–0.6 sulfhydryl group and thereafter almost no further loss of SH group occurred.

Purification and properties of pancreatic glutathione-insulin transhydrogenase.

An enzyme, similar if not identical to the glutathione-insulin transhydrogenase (thiol:protein disulfide oxidoreductase, EC 1.8.4.2) of beef liver, has been isolated and purified from beef pancreas. Evidence is presented that this pancreatic enzyme, as reported previously for the hepatic GSH-insulin transhydrogenase, catalyzes the reductive cleavage of the disulfide bonds of insulin. Some of its properties, including kinetic constants, are reported.