Characterization of a Rat Liver Protease with Specificity for Insulin1

Publisher Website
Google Scholar
Abstract
A soluble hepatic enzyme which preferentially cleaves insulin has been isolated from the 100,000 X g supernatant of rat liver. This enzyme was purified 97–fold by ultracentrifugation, Ca3(PO4)2 gel adsorption—elution, Sephadex G-200 filtration and DEAE-Sephadex A–50 chromatography. The enzyme was estimated to have a mol wt of 80,000 by its behavior on Sephadex G-200. The Km for insulin degradation was found to be 0.10μ.M. In fractionated liver homogenate, the 100,000 X g supernatant was found to be responsible for 96% of the total insulin degrading activity. The remaining 4% of activity was in the debris, mitochondrial and microsomal fractions. The enzyme exhibited a relatively narrow pH range with the optimum activity occurring at pH 7.6. The enzyme was shown to be proteolytic and independent of glutathione (GSH) for enzymatic activity. Marked specificity for insulin degradation was found as indicated by a 15–fold greater rate of insulin destruction over that for proinsulin. Human growth hormone was not appreciably degraded by the purified enzyme. The study of other proteolytic enzymes (trypsin, chymotrypsin, and papain) showed no specificity for insulin degradation over proinsulin. Mercaptoethanol and GSH slightly stimulated enzyme activity, whereas Nethylmaleimide and ρ-hydroxymercuribenzoate were shown to be potent inhibitors of enzymatic activity. These data suggest that a sulfhydryl group in the enzyme molecule is necessary for its activity. Tolbutamide and phenformin were found to inhibit the enzyme by a complex type of noncompetitive inhibition. These studies demonstrate the presence of an insulin protease in the liver and suggest that this enzyme is the major route for insulin catabolism in the body. (Endocrinology91: 633, 1972)

This publication has 0 references indexed in Scilit: