INSULIN AND GLUCAGON DEGRADATION BY KIDNEY .2. CHARACTERIZATION OF MECHANISMS AT NEUTRAL PH

Examination of insulin and glucagon degradation by rat kidney subcellular fractions revealed that most degrading activity was localized to the 100,000 .times. g pellet and 100,000 .times. g supernatant fractions. Further characterization of the degrading activities of the 100,000 .times. g pellet and supernatant suggested that 3 types of enzymatic activity were present at neutral pH. From the cytosol an enzyme with characteristics of the insulin glucagon protease of skeletal muscle was purified. This enzyme appeared to be responsible for insulin degradation by the kidney at physiological insulin concentrations. This enzyme also contributed to glucagon degradation but was not the most active mechanism for this. In the 100,000 .times. g pellet at least 2 separate enzymatic activities were present. One of these had properties consistent with those described for glutathione insulin transhydrogenase and appeared to be responsible for insulin degradation at high insulin concentration. The other enzyme was associated with the brush border and had properties consistent with the brush border neutral protease. This enzyme appeared responsible for glucagon degradation at both low and high substrate concentrations. An apparent marked synergism between the 100,000 .times. g pellet and the 100,000 .times. g supernatant was noted for insulin degradation at physiological insulin concentrations. Pellet glucagon-degrading activity and soluble insulin-degrading activity were necessary for this. The mechanism was limited insulin degradation by the soluble enzyme resulting in both trichloroacetic acid[TCA]-precipitable TCA-soluble fragments followed by further degradation of the fragments by the glucagon-degrading enzyme resulting in an additional increase in TCA-soluble products.