Substrate Studies for Insulin‐Specific Protease

Insulin‐specific protease, a soluble cellular enzyme from rat skeletal muscle which has been purified recently as a single enzyme, has been studied in regard to its substrate specificity, using various immunoreactive and biologically active insulin and proinsulin intermediates. The rate of degradation of pork insulin taken as 100% was compared to other insulin and proinsulin derivatives. Porcine proinsulin intermediates consisting of cleaved proinsulin, desdipeptide, desnonapeptide and destridecapeptide‐proinsulin, as well as desalanine, monoarginine and diarginine‐insulin, were degraded at 19.8, 25.6, 63.5, 73.7, 101.5, 98 and 98% of the activity of insulin, respectively. Rates of degradation of beef proinsulin, and intermediates I and II were 6, 20.8 and 5.9% of that of insulin with insulin‐specific protease, respectively. Studies of Km and V determinations of pork insulin and proinsulin and their intermediates revealed that all the substrates had similar V values (1.0 pmol/min); whereas the Km values (nM) were as follows: insulin, 22.2; desalanine‐insulin, 15.8; monoarginine‐insulin, 24.4; diarginine‐insulin, 24.4; proinsulin, 857.2; cleaved pro‐insulin, 234.2; desdipeptide‐proi