Substrate studies for insulin-specific protease.

Abstract

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‐proinsulin, 176.0; desnonapeptide‐proinsulin, 55; and destridecapep‐tideproinsulin, 44. Reduced proinsulin, labeled with iodo[14C]acetamide, did not show increased degradability by insulin‐specific protease as compared to native proinsulin. These studies suggest that one requirement for optimal substrate activity may be the deblocking of the amino end of the A chain of insulin. The blocking of the amino end, which is present in proinsulin or proinsulin intermediate, will reduce degradability of these substrates by insulin‐specific protease. The fact that the cleavage of disulfide bonds of proinsulin resulted in no further activation of proinsulin supports the above and indicates that steric hindrance may have a lesser role in the proinsulin molecule's inability to be degraded by insulin‐specific protease.