Cleavage of Bovine Insulin by Rat Adipose Tissue

Abstract

A previous study in this laboratory showed that rat adipose tissue contains an insoluble enzyme system which cleaves bovine insulin into numerous fragments soluble in 5% trichloroacetic acid. The present study investigated the nature of the cleavage process and the structure of the cleavage products. The insoluble fraction derived from 10-150 g of homogenized adipose tissue from the rat was washed 3 times with aqueous buffer to remove H20-soluble components, and incubated at pH 7.5 in sodium phosphate or ammonium acetate buffer with 100-1000 mg of bovine insulin. After various periods of incubation, the insoluble enzyme system was removed by centrifugation and filtration and the incubation filtrate was studied by chemical and chromatographic methods. These data showed that the disulfide and acid amide groups of insulin remained intact, and that internal and terminal peptide bonds were hydrolyzed. Eleven free amino acids and 20 peptides produced by these hydrolyses were isolated by gradient elution from phosphocellulose columns, followed by 1-dimensional paper chromatography. The quantities of the various free amino acids were measured by the amino acid analyzer. The amino acids present in each peptide were identified by 2-dimensional paper chromatography of an acid hydrolysate of the peptide; from the amino acid composition the structure of the peptide could be deduced. The quantitative data on the composition of the mixture of free amino acids, and the qualitative data on the amino acid composition of the peptides, which appear during the cleavage process, are compatible with the following hypothesis: that the initial event is hydrolysis of internal bonds in the region of A13-14, A18-19, B11-12, B15-16, B24-25 and B25-26; and that the 5 or 6 peptides thus formed then undergo stepwise removal of terminal residues from both N and C termini. The insulin-cleaving enzyme system hydrolyzed synthetic dipeptides which are susceptible to aminopeptidase or carboxypeptidase action, confirming the presence of 2 nonspecific exopeptidase activities in the insoluble fraction of the rat''s adipose tissue; a variety of endopeptidase substrates other than insulin were not hydrolyzed, however, suggesting that the tissue enzyme which performs the initial internal hydrolyses may be specific for insulin.