Some problems of enzymatic assay of insulin degrading enzyme extracted from pig skeletal muscle were described.
Enzymatic assay of various kinds of insulin degrading enzyme has been generally carried out by determinig the131I-labeled degradation product of 131I-insuliinn trichloroacetic acid soluble supernatant (TCA method).As determined by this method, degradation appeared to level off when approximately 60% of the 131I-insulihna d been degraded, and so the enzyme activity appeared to be considerably lower than it was. This seemed to be due to the fact that a portion of the TCA soluble degradation products was carried down with the protein which was precipitated in the incubation mixtures by TCA.However, the relative rate and extent of degradation under various conditions was reliably determined with good reproducibility and simplicity by this method.
On the other hand, non-degraded insulin was assayed by double antibody radioimmunoassay after terminating the enzyme reaction by N-ethylmaleimide (RIA method). As determined by this RIA method, the degradation of insulin appeared to proceed more rapidly to completion, and so the enzyme activity proved to have been able to be determined more correctly than TCA method.
In conclusion, it seems that there is no perfect method of enzymatic assay of insulin degrading enzyme, though both TCA method and also RIA method have their advantages and disadvantages.Therefore, either, TCA method or RIA method should be selected as the experimental design.
