Sequential Degradation of Insulin by Rat Liver Homogenates

Abstract

Insulin was incubated with rat liver homogenate in the presence of glutathione. The products formed were examined by chromatography on a Sephadex G-75 column, with 50% acetic acid as eluent. The results show that insulin is degraded by rat liver homogenates in sequential order: first, a splitting of insulin into A and B chains by glutathione-insulin transhydrogenase, followed by proteolysis of the resulting polypeptides to small molecular weight components.Insulin is rapidly inactivated in vivo (1-3) and in vitro (4-6). Inactivation of insulin could take place by cleavage of insulin with glutathione(GSH)-insulin transhydrogenase (insulin transhydrogenase) (7-11), and/or by proteolysis with wellknown proteolytic enzymes (trypsin, chymotrypsin, exopeptidase) or with insulin-specific proteolytic enzymes. A and B chains of insulin are degraded by proteolytic enzymes more rapidly than is native insulin (12). It is conceivable that the inactivation of insulin takes place in a step-wise manner; the first step might be the cleavage of insulin by the transhydrogenase, followed by hydrolysis of the resulting A and B chains. This would suggest a key role for insulin transhydrogenase in the disposal (i.e., metabolism) of insulin. However, no direct experimental evidence supports these ideas.The experiments reported in this paper indicate that insulin is degraded in a step-wise manner. MATERIALS AND METHODSGSH-insulin transhydrogenase was prepared from rat liver in a highly purified state according to the method of Tomizawa and Halsey (13); the same procedure has been used for the isolation of purified transhydrogenases from beef liver (13), beef pancreas (10), human liver (9), and human kidney (11). Antiserum to the purified rat liver enzyme was produced in rabbits by a procedure used previously to produce antibodies to beef pancreatic and human liver transhydrogenases (14). In an Ouchterlony double-diffusion test with antibody, the rat enzyme showed a single precipitin band. The hr, rats were decapitated with a miniature guillotine (Harvard Apparatus Co.) and as much blood as possible was drained from the carcass. The livers were removed, rinsed with cold tap water, and placed in ice-cold beakers. A 1-g piece of liver was mixed with 4 ml of 0.25 M sucrose-5 mM EDTA (pH 7.5), and another 1-g piece was mixed with Krebs-Ringer bicarbonate buffer. The pieces were then homogenized for 40 sec with a Polytron PT-20/2 homogenizer at "position 10" of the rheostat, which was arbitrarily divided into 30 equal parts from the off position to fully on. During homogenization, the homogenization tube was kept cold. The homogenates were used without any further treatment; this procedure was chosen to minimize the role of diffusion processes (3), and at the same time retain the activity of all the liver proteins.Immunologic Procedures. These were done as described (14). In a series of tubes, different volumes of homogenate were mixed with 0.05 ml of antiserum to insulin transhydrogenase from rat liver. The final volume in each tub...

Chloroplast Glutathione Reductase

“…GSH has been implicated in a large number of reactions including the nonenzymic reduction of thial groups, enzymic reduction of disulfide bridges of protein with consequent deactivation (10) or activation of the protein, and as a substitute for GSH peroxide (12).…”

Intracellular Carboxy‐Terminal Degradation of the αA Chain of α‐Crystallin

“…If indeed the degradation of the aA chain is an enzymatic process, it must be caused either by an exopeptidase with carboxypeptidase C-like activity (which is as yet not known to occur in mammalians), or by a specific endopeptidase. Such a protein-specific protease is known to be responsible for the sequential degradation of insulin [23,24]. Other proteins appear to be degraded by endogenous proteolytic activity, while being unable to degrade exogenous protein substrates [25].…”

How many distinct enzymes are responsible for the several cellular processes involving thiol:protein‐disulphide interchange?

“…Studies on the metabolism of polypeptide hormones, especially insulin, have indicated that reduction of their disulphide bonds plays an important part in their degradation [1][2][3][4][5][6][7]. Studies on insulin degradation using whole cells [7], homogenates [1][2][3]5,6] and sub-cellular fractions [4,8] show that the reduced A chain and various ill-characterised oligomers of the A and B chains are major products.…”

“…Studies on insulin degradation using whole cells [7], homogenates [1][2][3]5,6] and sub-cellular fractions [4,8] show that the reduced A chain and various ill-characterised oligomers of the A and B chains are major products. Considerable evidence has been advanced showing that a two-step pathway of degradationreduction followed by proteolysis -occurs at both high and at physiological insulin concentrations [5][6][7].…”

“…Essentially two activities have been used for their analysis namely the reduction of insulin by GSH, to yield GSSG and reduced A and B chains [1][2][3][4][5][6][7][8] a type (1) reaction, and the thiol-dependent regain of enzyme activity from fully oxidised incorrectly disulphide-bonded ribonuclease ('scrambled' ribonuclease), a reaction of type (4) [64][65][66][67][68][69][70][71][72][73][74][75]. These two activities have been formally named glutathione: protein-disulphide (insulin) oxidoreductase (EC 1.8.4.2) and protein-disulphide isomerase (EC 5.3.4.1), respectively.…”