Insulin-degrading enzyme is capable of degrading receptor-bound insulin.
In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.
Yonezawa
K
K
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Yokono
K
K
Shii
K
K
Hari
J
J
Yaso
S
S
Amano
K
K
Sakamoto
T
T
Kawase
Y
Y
Akiyama
H
H
Nagata
M
M
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000378:metabolism*; D007339:Insulysin / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D013379:Substrate Specificity
D007328:Insulin; D011972:Receptor, Insulin; D010447:Peptide Hydrolases; D007339:Insulysin