Studies on Purification and Characterization of Insulin Degrading Enzyme Extracted from Rat, Rabbit and Pig Muscles

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Liver, kidney, muscle and adipose tissue appear to be the major site of insulin degradation. Mirsky and Broh-Kahn (1949) investigated the insulin inactivating capacity of various tissues and postulated that insulinase might be responsible for insulin degradation. Recently, Brush, Cheng and co-workers have indicated that a soluble enzyme extracted from rat skeletal muscle rapidly degrade insulin and is of a remarkable degree of specificity for the insulin molecule. We have carried out further experiments in order to verify the new enzyme extracted from rabbit, rat and pig muscles. In order to facilitate the procedure of enzyme purification. The Brush's method was modified. Namely the rear leg muscles of rat, rabbit and pig were removed, minced, and then homogenized at a high speed in 0.35 M sucrose (5 ml/gm tissue). The homogenate was prepared by filteraid filtration at 4° and the supernatant was dialysed three times for at least 4 hours, each time aganist 20 volumes of distilled deionated water. After adjusting with acetic anhydride the dialysed supernatant to pH 5, a sufficient amount of 0.015 M acetate buffer, pH 5, was added to bring it up to 6.5 L. Ca3 (PO4)2 gel in a sufficient amount was added to obtain a ratio of gel to protein of 0.28: 1. The gel-protein mixture was stirred at 4° for 20 minutes, followed by centrifugation at 10,000×g for 10 minutes. The supernatant was discarded and the gel resuspended and stirred for 20 minutes in 0.05M phosphate buffer, pH 6.2. Following sedimetnation of the gel by centrifugation, it was resuspended and stirred for 20 minutes in 0.05 M phosphate buffer, pH 7.5. Removal of the gel was accomplished by centrifugation, and the supernatant containing the enzyme was rapidly frozen at-70° for eventual use. Furthermore, the supernatant, from which the enzyme had been extracted, was washed by 0.05M phosphate buffer, pH 7.5, 4 times. The final protein content was obtained about 1, 000 mg from 1.5kg of muscle. Enzyme assay was carried out by the modified Mirsky's method. And also the rates of degradation were assayed for remaining immunoreactive insulin according to the method of Morgan and Lazarow. A marked activity was found in each of pig and rat muscle, i.e. specific activity of 2.29-2.56μU/mg of the enzyme protein. The enzyme from rabbit muscle, however, showed no remarkable activity. The maximum activity was obtained at pH 8.0-8.6, declining on each side of this optimum range. The activity of the enzyme was blocked by N-ethylmaleimide, DFP and boiling treatment. The inhibitors of trypsin and chymotrypsin, however, did not impair the enzymatic activity. Furthermore, the rate of degradation of proinsulin attacked by the enzyme was from 0 to 18%, while that of insulin was 100%. It was also found to be a competitive inhibitor of insulin degradation. Km for insulin degradation, as indicated, was 1.23μM, and K1 for proinsulin was 2.06μM. These results suggest that the enzyme is a sulfhydryl dependent protease which is of quite similar characteristics to Brush's one and has a species specificity for insulin degradation. Through this purification process, it appears that the enzyme-inhibitor most probably exists.
Japan Society of Clinical Chemistryの論文

Studies on Purification and Characterization of Insulin Degrading Enzyme Extracted from Rat, Rabbit and Pig Muscles

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