Identification of γ-Endorphin-Generating Enzyme as Insulin-Degrading Enzyme

Abstract

The EL-4 thymoma cell line contains a peptidase which converts beta-endorphin to beta-endorphin 1-17 (gamma-endorphin), beta-endorphin 1-18, and their corresponding C-terminal fragments. This enzyme was purified approximately 700-fold to a single band on an SDS-polyacrylamide gel (106 kDa) in 16% yield. Estimation of the native molecular weight by molecular sieve chromatography gave a value of approximately 220 kDa, indicating that this enzyme is a dimer. Peptide sequencing demonstrated this activity can be attributed to insulin degrading enzyme, a previously described member of the inverzincin family (Hooper, 1994). Kinetic studies with a number of peptide substrates indicate that the enzyme preferentially cleaves on the amino side of hydrophobic or basic residues. However, the substrate specificity is more complex since not all basic and hydrophobic residues in a peptide are cleaved. The enzyme exhibits a requirement for a P'2 residue. On the basis of kcat/K(m) values, insulin, growth hormone releasing factor, and beta-endorphin are nearly equivalent substrates for the enzyme; however, growth hormone releasing factor and beta-endorphin exhibit a 40-fold higher kcat, but a 10-fold decreased affinity relative to insulin. A role for insulin-degrading enzyme as both a beta-endorphin-processing and -inactivating enzyme is implicated from these studies.

Insulin-Degrading Enzyme as a Downstream Target of Insulin Receptor Signaling Cascade: Implications for Alzheimer's Disease Intervention

“…Also, IDE is not only a proteolytic degrading enzyme for insulin and A␤, but also for a number of other peptides such as glucagon (Kirschner and Goldberg, 1983), ␤-endorphin, and dynorphins (Safavi et al, 1996). It is interesting to note that both AD and Down syndrome patients show significant elevations of dynorphins in the frontal cortex tissue (Risser et al, 1996), suggesting that IDE correction by insulin signaling in AD could also bring down other substrates that are abnormally high.…”

Anion Activation Site of Insulin-degrading Enzyme

“…More recently, IDE has received considerable attention for its role in degrading amyloid ␤ (A␤) peptides (4 -7), and a genetic link between IDE and late onset Alzheimer disease has been reported (8). IDE degrades a number of other physiological peptides in vitro, including IGF-1 and IGF-2 (9), glucagon (10), atrial natriuretic peptide, TGF-␣ (11), and ␥-endorphin (11). In addition to its metabolic role in peptide degradation, IDE has been reported to play a role in the inhibition of proteasome function by insulin (12), to degrade oxidized proteins in peroxisomes (13), to serve as a receptor accessory factor that enhances androgen and glucocorticoid receptor binding to DNA (14), and to serve as a receptor for the Varicella-Zoster virus (15,16).…”

“…IDE exists as an equilibrium mixture of monomers, dimers, and tetramers, with dimers thought to be the predominant and most active state (11,20). IDE is unusual among zinc metallopeptidases in that it exhibits allosteric kinetic behavior, with small peptide substrates increasing the activity of the enzyme toward the same or other small peptides (20).…”

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

“…Many members of the family of metalloproteases have been implicated in extracellular processing of neuropeptides. Endopeptidase 24.15, insulin-degrading enzyme, and Nardilysin have been shown to cleave various opioid peptides and other bioactive pentapeptide (Chu and Orlowski, 1985;Chesneau et al, 1994;Safavi et al, 1996). Thus, it appears that members of diverse families of processing enzymes are involved in the processing of neuropeptide precursors at various processing sites.…”