PREPARATION AND CHARACTERIZATION OF NOVEL SUBSTRATES OF INSULIN PROTEINASE (EC-3.4.99.45)

The specificity of insulin proteinase (EC 3.4.99.45) has been difficul t to categorize using only its natural substrates. By exploiting the f act that two substrates competing for the same enzyme inhibit one anot her, we have found some new substrates of the insulin proteinase from porcine muscle. Two of these substrates, a tryptic fragment of BSA and a fragment of cytochrome c, have been shown to be cleaved at a single site. The albumin fragment, as well as another fragment of cytochrome c,have suceptibilities (V-max./K-m) comparable with that of insulin. In a second aspect of the study, the porcine-muscle enzyme was shown t o be related to other members of its superfamily in that it was immuno precipitated by a monoclonal antibody raised against the insulin-degra ding enzyme from human red blood cells and has the same cleavage sites on insulin as has the rat skeletal-muscle insulin proteinase. We note , however, a possible discrepancy between our results and those of ano ther group regarding the subunit size (110 kDa) of the immunoprecipita ted material.