Topology of glutathione-insulin transhydrogenase in rat liver microsomes

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent ('inactive') state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A2 and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A2 resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A2 is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of the endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.