INVIVO ASSOCIATION OF [I-125] INSULIN WITH A CYTOSOLIC INSULIN-DEGRADING ENZYME - DETECTION BY COVALENT CROSS-LINKING AND IMMUNOPRECIPITATION WITH A MONOCLONAL-ANTIBODY

A cytosolic insulin-degrading enzyme (Mr = 110,000) was found to be cross-linked to [125I]-insulin in intact human hepatoma cells, HepG2, incubated with the hormone and treated with the bifunctional cross-linker, disuccinimidyl suberate. The labeling of this protein was greatly increased by concurrent treatment of the cells with N-ethylmaleimide, to the extent that the amount of [125I]-insulin cross-linked to the enzyme in these cells was approximately 20 to 50% that cross-linked to the insulin receptor. The labeling of the insulin-degrading enzyme required the prior interaction of [125I]-insulin with its receptor as well as a temperature- and energy-dependent processing of the hormone. The present work therefore supports a role for this protease in the cellular processing of insulin.