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A soluble enzyme partially purified from rat muscle is described which degrades insulin proteolylically with a high degree of specificity. The enzyme attacked proinsulin and proinsulin intermediates at only 3 per cent and 10 per cent of the rate with insulin, respectively. Proinsulin competitively inhibited insulin degradation with an inhibition constant of 0.28 JJM, whereas the K m for insulin was 0.18 /J.M. Insulin derivatives with amino or carboxyl terminal residues removed were attacked with K m 's about five times greater. Removal of an octapeptide from the B chain of insulin resulted in a K m the same as for insulin. The enzyme is inhibited by the sulfhydryl reagents, N-ethylmaleimide and p-hydroxymercuribenzoate, but not by an inhibitor of the pancreatic proteases, phenylmethylsulfonyl fluoride. The enzymatic activity was not inhibited by a large excess of a number of other proteins and polypeptide hormones. These results suggest that the enzyme is a sulfhydryldependent protease which is quite specific for insulin, parameters studied. DIABETES 20: 151-55, March, 1971. Although insulin degradation in rat liver by proteolysis 1 -2 or reductive cleavage 3 -4 has been described, the specificity of the first reaction has not been examined, and the second is very broad. 5 Previous work from this laboratory has indicated that a soluble enzyme from rat diaphragm 6 and from rat skeletal muscle 7 rapidly degrades insulin at physiological concentrations. This communication gives additional data on the properties and mode of action of the enzyme, together with evidence suggesting it possesses a high degree of specificity for the insulin molecule.

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A B S T R A C T A method has been described for the direct measurement of proinsulin in human plasma. The method makes use of an insulin-degrading enzyme designated "insulin-specific protease (ISP)", which is obtained from rat skeletal muscle. Under the conditions used, this enzyme rapidly degrades insulin and insulinlike polypeptides to nonimmunoassayable components, whereas proinsulin and proinsulin cleaved at position B5 ,s are not appreciably affected. Proinsulin responses were studied in four normal subjects and one patient with an insulinoma after a glucose load. Fasting proinsulin levels varied widely in the normal subjects, and the levels rose more slowly than TIR levels after glucose. IRP levels in the patient with an insulinoma were very high and fell to normal after re-

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