Purification and Characterization of a Protease with Specificity for Insulin from Rat Muscle

Abstract

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.

Purification of Insulin-Specific Protease by Affinity Chromatography

“…Insulin-degrading activity was demonstrated in the soluble fraction of cells from rat diaphragms (10). Brush partially purified the rat skeletalmuscle enzyme and demonstrated that this sulfhydryl enzyme proteolytically degraded insulin, but not proinsulin (11). A similar enzyme has been demonstrated in other rat tissues (12,13), and a similar enzyme in the liver has been partially purified (7).…”

“…After centrifugation, the supernatant and precipitate were counted in a Packard autogamma spectrometer. One unit of enzyme activity is that amount of enzyme that converts 1 fmol of insulin to acid-soluble material per min (11).…”

“…ISP in pH 7.5 buffer was incubated for 20 hr at 370 with 1.5 mg of porcine insulin (11). Similarly prepared enzyme was also incubated with 1.5 mg of bovine-serum albumin under the same conditions.…”

Insulin degrading enzyme activity and insulin binding of erythrocytes in normal subjects and Type 2 (non-insulin-dependent) diabetic patients

“…IDEA, purified to homogeneity, has been characterized recently as a sulphydryl-dependent protease, which specifically degrades insulin at physiological hormone concentrations into peptides smaller than the A-chain of insulin and can be inhibited by several protease inhibitors [14,15]. Though red blood cells are not a known target tissue of insulin action, they do carry insulin receptors, and erythrocyte IDEA appears to show properties similar to the insulin degrading proteases of insulinsensitive tissues from animal sources [1][2][3][4][5]10] and especially similar to the enzyme purified from human skeletal muscle [20].…”

“…A cytosolic soluble enzyme which rapidly degrades insulin by proteolysis has been found in many animal [1][2][3][4][5][6][7][8][9][10][11][12] and some human tissues [12][13][14][15][16][17][18][19][20]; reductive disulphide cleavage of insulin, i. e. a glutathione-insulin-transhydrogenase activity, has also been described [11,[20][21][22]. Evidence has accumulated that both insulin action and degradation might be mediated through binding of insulin to specific receptors on the plasma cell membrane as the initial step [12,13,18,19,23,24], although the precise nature of this relationship remains to be elucidated.…”