Insulin-degrading Activity of Plasma Membranes from Rat Skeletal Muscle: Its Isolation, Characterization, and Biologic Significance

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Abstract
We isolated the plasma membrane from rat skeletal muscle without using drastic procedures such as extraction by salts or other agents. As a result of the purity of our preparations, evaluated by enzymatic markers, lipid composition, 125l-insulin specific binding, and morphologic examinations, we were able to use the plasma membrane to study insulin degradation. Isolated plasma membranes were capable of degrading insulin, but 95% of total degrading activity was found in the cytosol fraction. The membranes proteolytically degraded the hormone with a high degree of specificity and a pH optimum of 7.0. The extent of degradation depended on time, temperature, and protein concentration. The apparent Km for insulin was 1.7 × 10−7 M. N-ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB) markedly inhibited insulin degradation by membranes, whereas glutathione (GSH) and dithíothreitol (DTT) were stimulatory. These characteristics of insulin-degrading activity in the plasma membrane (membrane-IDE) were similar to those of the partially purified insulin-degrading enzyme from the cytosol fraction (cytosol-IDE). To clarify the biologic significance of IDE, we examined the cytosol- and membrane-IDE activities under various conditions in rats grouped as follows: fed, fasted for 1 day, fasted for 3 days, refed, refed + actinomycin D, diabetic, and hyperinsulinemic (insulinoma). A positive correlation with a high coefficient (r = 0.674, P < 0.001) was demonstrated between cytosol-IDE and the plasma insulin concentration but not between membrane-IDE and insulin levels. While actinomycin D had no effect on membrane-IDE levels, it abolished the insulin-mediated rise in cytosol-IDE activity, suggesting that the latter was dependent on RNA synthesis.