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 sulfhydryldisulfide 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 N-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 second having an Mr of 67Kd. Immunoblots using polyclonal antibodies revealed an additional major immunoreactive band of 32Kd in the cow lens epithelial layer as well as in the isolated cortical and nuclear portions. Rat lenses showed no immunoreactive 32Kd band. Using a bovine cortical/nuclear fraction the 32Kd reactivity was found to be associated with the beta H-crystallin fraction, but the extract failed to show GIT activity with the insulin substrate. This suggests that beta H-crystallin may share a common epitope with GIT.
Darrow
R M
RM
Department of Biochemistry, School of Medicine, Wright State University, Dayton, OH 45435.
Morris
J I
JI
Organisciak
D T
DT
Varandani
P T
PT
D000818:Animals; D002417:Cattle; D015151:Immunoblotting; D007903:Lens Capsule, Crystalline / Q000201:enzymology / Q000648:ultrastructure; D007908:Lens, Crystalline / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione) / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D014018:Tissue Distribution
D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione)