Endocrinology, Vol 118, 1989-1996, Copyright © 1986 by Endocrine Society

ARTICLES

Degradation of insulin by insulin-degrading enzyme and biological characteristics of its fragments

K Yonezawa, K Yokono, S Yaso, J Hari, K Amano, Y Kawase, T Sakamoto, K Shii, Y Imamura and S Baba

We have previously reported on the purification, characterization, and biological significance of insulin-degrading enzyme (IDE) from pig and rat skeletal muscle. In the present study, we have investigated the detection and the HPLC separation of degradation products of native insulin from the reaction of monocomponent porcine insulin with affinity-purified pig IDE. Insulin was degraded by IDE in a time- and dose-dependent manner. Eight peaks (peaks I through VIII) appeared after 1 h of incubation, and peak V was identified as insulin. Among seven peaks representing degradation products, peak VI appeared most rapidly at 30 sec of incubation, increased until 10 min, and then decreased after 15 min of incubation; and six degradation products other than peak VI were not detected within 15 min of incubation, suggesting that peak VI was an initial degradation product of insulin produced by IDE and converted into relatively low molecular weight products as incubation time increased. The generation of peak VI may be due to cleavage at a peptide bond between the interchain disulfide bonds of the A or B chain. Subsequently, the split insulin derivative (peak VI) was evidently further degraded to relatively low molecular weight intermediates, such as peaks III and IV, peaks II and VIII, or peaks I and VII, because these pairs of peaks appeared and were degraded concomitantly. The peptide products designated as peaks IV, VI, VII, and VIII had both immunoprecipitability by antiinsulin antibodies and binding capacity to IM-9 lymphocytes, whereas the less hydrophobic intermediates (peaks I, II, and III) did not have these activities. Since some of these peptides have insulin-like properties, amino acid analysis of these products may enable us to identify not only the splitting position of insulin by IDE but also the site of the hormone for receptor binding.