The occurrence of glutathione—insulin transhydrogenase (protein—disulfide interchange enzyme) in the lens

Glutathione-insulin transhydrogenase (GIT, thiol:protein-disulfide isomerase/oxidoreductase, E.C. 5.3.4.1/1.8.4.2) catalyzes via sulfhydryl-disulfide interchange, the scission as well as formation of disulfide bonds in many diverse proteins. Using insulin as a substrate, the lens epithelial layer of cows, rats and rabbits was found to contain GIT activity. The enzyme's activity is activated by GSH and inhibited by M-ethylmaleimide. Subcellular distribution of bovine lens epithelial homogenates showed that the majority of GIT activity is located in the insoluble fraction (10,000 g pellet) and in the high molecular weight fraction (60,000 g pellet). Lens epithelial extracts were subjected to SDS-PAGE followed by Western blot, and probed with a polyclonal antibody to rat liver GIT, or with either of two monoclonal antibodies directed against different epitopes of the enzyme. Lens epithelium was found to contain two forms of GIT, one with the same molecular weight as the purified enzyme (Mr 56Kd), and a secon...