Amos Neidle

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.

Histones: Species and Tissue Specificity

Within a given species (rat, mouse, guinea pig, rabbit) the histones of brain, liver, and kidney are indistinguishable on disc electrophoresis in polyacrylamide gels. However, characteristic species differences were observed. In the immature rat, the histones of brain and liver, while very similar to each other, differ from those of the adult. The histones of the thymus of the immature and adult rat exhibited a pattern corresponding to that of the immature brain and liver.

Chemical stability and metabolic utilization of asparagine peptides

Abstract l -Leucyl- l -asparagine and l -asparaginyl- l -leucine are utilized as well as l -asparaginylglycine and glycyl- l -asparagine by Leuc. mesenteroides . The growth response to these peptides approximates that to l -aspartic acid and is far superior to that to l -asparagine. l -Aspartylglycine and glycyl- l -aspartic acid at high concentrations are superior to l -aspartic acid as a source of the dicarboxylic acid. The glycine peptides serve as better sources of glycine than glycine itself. The growth response to the leucine peptides is equal to that to leucine. Cell-free extracts of Leuc. mesenteroides show strong asparaginase activity. Observations on the enzymatic splitting of the asparagine peptides suggest that the peptides are cleaved into the amide and glycine or leucine and that the free amide is deamidated. It appears that the superior utilization of asparagine peptides when compared with that of asparagine is the result of the inability of the latter to reach the sites of its metabolism by the bacterial cell. Procedures for the syntheses of leucylasparagine, asparaginylleucine, α-aspartylglycine and glycylaspartic acid are described. The instability of glycylasparagine in acid and of asparaginylglycine in alkali is demonstrated and discussed.