A New Determination of Insulin-degrading Enzyme by Radioimmunoassay

Abstract

We have reported that insulin-degrading enzyme (IDE), which proteolytically degrades insulin with a high degree of specificity, is involved in insulin metabolism in the body. A radioimmunoassay for IDE has been developed using antiserum raised in a rabbit to purified pork muscle IDE. The antibody cross-reacted with rat, pork and human IDE.Pork muscle IDE purified by affinity chromatography showed a 3, 000-fold purification in terms of its activity and a single band on polyacrylamide gel electrophoresis (PAGE).Purification of ¹²⁵I-IDE was accomplished by gel filtration on Sepliadex G-50, G-200 and PAGE.Rat kidney and liver extracts revealed parallel dilution curves, and no significant cross-reaction was observed with either trypsin, amylase or cathepsin C purified from spleen lysosomes.The sensitivity cf the assay ranged from 10 to 500 ng/ml, and the intra-and interassay coefficients of variation were 6 and 11%, respectively.There was some discrepancy between the concentration of IDE measured by RIA and insulin-degrading activity among liver, kidney and muscle extracts.This discrepancy was probably due to miscellaneous factors responsible for insulin degradation included in the tissue extracts, especially in the liver.In sera from normal and IN1DDM subjects, IDE was not detectable in our RIA.One young female, who was resistant to se and im insulin but responsive to a mixture of aprotinin (a protease inhibitor) or iv insulin, however, had an IDE level of 15 to 25 ng/ml.In this case, IDE could play a major role in insulin resistance. The present RIA, therefore, may offer a useful tool for measuring the true concentration of IDE under varius experimental conditions.