Metabolism of insulin and glucagon. Breakdown of radioiodinated insulin and glucagon in rat liver cell fractions.

Abstract

An enzyme catalyzing the reductive breakdown of insulin was isolated and purified from microsomes of rat liver. Its properties resemble strongly those of glutathione-insulin transhydrogenase (thiol-protein disulfide oxidoreductase) from bovine liver, human liver, bovine pancreas, and human kidney. The enzyme was separated from microsomes by deoxycholate and purified by gel filtration on Sephadex G-75 and subsequent chromatography on DEAE-Sephadex A-50. Disc electrophoreses showed that highly purified, fresh enzyme preparations consist of only two enzymatically active proteins, forming one main band and a faint band, without any further visible contaminants. The content of glutathione-insulin transhydrogenase in rat liver amounts to about 0.5–1% of total liver proteins as calculated from the purification factor. The molecular weight of the microsomal transhydrogenase determined by gel filtration on Sephadex G-75 is 50000 to 55000. Using disc electrophoresis in dodecylsulfate-containing gels molecular weights of 60000 and 121000 were determined for the main component and the second, slower-migrating band of the enzyme, respectively. The Michaelis-Menten constants for insulin and glutathione (in the presence of EDTA) are 31 μM (mol. wt 6000) and 1.43 mM, respectively. Maximum breakdown of 125I-labelled insulin was found at pH 7.3. It seems very probable that our transhydrogenase from rat liver microsomes is identical or very similar to the enzyme catalyzing the reactivation of “randomly-oxidized” ribonuclease (Anfinsen).