Studies on the specificity of an insulin-inactivating system of the liver.

In a previous report (5), evidence was presented indicating that insulin is probably proteolytically inactivated by a system in rat liver extract. In the course of these studies, it was found that, in contrast to the degradation which occurs with insulin-P31, incubation of human serum albumin-P31 or ribonuclease-1131 with liver extract resulted in no apparent degradation; that is, with either substance there was a negligible rise in radioactivity soluble in trichloroacetic acid (TCA). This observation suggested the possibility that all proteins might not be rapidly attacked by this liver system. Use of only P31-labeled proteins in studying substrate specificity has an obvious limitation. This is the possibility that cleavage may not necessarily give rise to TCA-soluble radioactivity. The question of specificity was therefore investigated by the technique of substrate competition in which the effect of various proteins upon the extent of degradation of insulin-I131 was measured. Also, the increase in ultraviolet absorption at 280 rnp or in the nitrogen content of the TCA-soluble fraction was determined after incubation of certain non-labeled proteins with a liver preparation. The studies described in this report were carried out with rat and beef liver extracts and a partially purified enzyme preparation from the latter.