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 1000/, 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 9S0/, of the activity of insulin, respectively.Rates of degradation of beef proinsulin, and intermediates I and IS were 6, 20. 8 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.Two systems for degradation of insulin have been reported in the literature: 1. insulinase, a system proposed by Mirsky, which degrades insulin through the hydrolytic cleavage of the peptide bonds. This system was shown to have high specificity for insulin since it did not degrade other proteins such as albu- mined that the glutathione-dependent enzyme is located in the microsomes ; whereas, the glutathioneindependent enzyme is located in the cytosol fraction.Demonstration of proinsulin as the precursor of insulin its intermediates in circulation [9, lo], prompted us to evaluate the relative suitability of these intermediates for insulin-degrading enzyme systems. The earliest work reported on the comparative ability of these enzymes in regard to substrate specificity was that of Brush [ll] in 1971, demonstrating that the supernatant fraction from a centrifugation of muscle homogenate a t 100000 x g can degrade insulin 30 times more than proinsulin. This enzyme, which is called insulin-specific protease, has been demonstrated in all organs of rats [12] and has been purified in muscle [8], kidney [12] and liver [13] in our laboratories, showing that greater than goo/, degradation of insulin is accomplished with insulinspecific protease, not glutathione insulin transhydrogenase. Furthermore, with the use of affinity chromatography, insulin-specific protease has been purified from muscle of rat and has been shown by gel electrophoresis to be one single band. Insulinspecific protease is highly specific for insulin with a

Insulin Metabolism and Degradation*

“…Although proinsulin was degraded very slowly, compounds intermediate between proinsulin and insulin were degraded more readily, with those more like insulin degraded most rapidly (102). A strong correlation between biological activity (in the fat cell assay) and susceptibility to degradation by the enzyme was found, confirming that the enzyme recognized the insulin structure with considerable specificity, just as the insulin receptor does (103).…”

“…Insulin derivatives that have decreased biological activity also appear to have a decreased susceptibility to degradation, and in many cases the amount of decrease in the two properties is similar (102,103,125). For example, the susceptibility of a group of proinsulin intermediates to degradation with insulin protease correlated very well with their biological activity in the isolated fat cell.…”

In vitro activity of biosynthetic human diarginylinsulin

“…The first step of insulin degradation may be mediated by a "insulin-specific protease" [12]. No difference in substrate activity of an insulin-specific protease was reported for porcine diarginylinsulin and insulin, the ability of the enzyme to degrade proinsulin, however, was negligible [39]. In our study the degradation of 125I-proinsulin was reduced in comparison to diarginylinsulin and native in-S. Zeuzem et al: Human diarginylinsulin sulin.…”

Inhibition of insulin degradation by nonsuppressible insulin-like activity.

“…This enzyme is inhibited by plasma (17), and proinsulin intermediates (18) but not by a number of other peptides and proteins which have been examined (8,15).…”