Donald D. Clarke

The incorporation of amines into protein

Abstract 1. 1. A Ca++-activated enzyme system derived from the soluble fraction of guinea pig liver which incorporates a wide variety of amines into proteins is described. 2. 2. The incorporation of amine is independent of an extraneous source of energy. 3. 3. The reaction system is shown to consist of an amine-accepting protein substrate and an enzyme. Various purified proteins can serve as substrates in addition to guinea pig liver supernatant protein. 4. 4. Factors which influence the extent of amine incorporation, such as pH, temperature, amine concentration, inhibitors, and activators, are reported. 5. 5. Possible mechanisms for the reaction and the relation of this system to other amine-incorporating reactions are discussed.

Amine incorporation into insulin as catalyzed by transglutaminase

Abstract Since insulin serves as an excellent substrate in the enzymically catalyzed amine incorporation reaction, a detailed study of its substrate properties was undertaken. A method for the separation of the A and B chains of oxidized insulin employing Dowex 50 resin is described. Native zinc-free insulin inhibited the enzyme, an inhibition which could be overcome by the addition of glutathione or other reducing agents. The mechanism of the reaction with insulin as substrate involves a replacement of the amide groups of peptide-bound glutamine but not of asparagine by the amine. The incorporated amines (N 15 H 3 , methylamine, hydroxylamine) are localized solely in the A chain of insulin. Isolated A chain but not B chain can serve as an amine acceptor. The use of hydroxylamine as a replacing amine permits the measurement of the reactivity of acid-soluble peptides and proteins readily by determining the extent of hydroxylamine incorporation colorimetrically. In the presence of added amine, the ammonia equivalents released correspond to those of amine incorporated at the pH optimum. In the absence of added amine, cross-linking of the protein-bound lysine accounted only for a small portion of the liberated ammonia. The name transglutaminase is suggested for the enzymic activity catalyzing amine incorporation.

Liver transglutaminase activity under various experimental conditions

Bacterial endotoxins and Haemophilus pertussis vaccine were found to increase the activity of liver transglutaminase in mice, rats, and guinea pigs. Enzyme activity increased also in the spleen of the injected mice, whereas no significant changes were found in the thymus, the lung, and the brain. The enzyme activity in adrenalectomized mice increased in the liver. Pretreatment of the animal with cortisone antagonized the rise in liver transglutaminase after endotoxin administration. The enzyme obtained from the liver of endotoxin-treated mice has the characteristics of transglutaminase obtained from the livers of normal mice. Mice and rats have a low enzyme activity in the liver and show a considerable increase upon administration of H. pertussis vaccine and purified endotoxins. Guinea pig liver, which is the richest source of transglutaminase so far found, shows a significant increase only upon the administration of high doses of endotoxins but not of pertussis vaccine.