IDENTIFICATION OF GAMMA-ENDORPHIN-GENERATING ENZYME AS INSULIN-DEGRADING ENZYME

The EL-4 thymoma cell line contains a peptidase which converts beta-en dorphin to beta-endorphin 1-17 (gamma-endorphin), beta-endorphin 1-18, and their corresponding C-terminal fragments. This enzyme was purifie d 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 similar to 220 kDa, ind icating that this enzyme is a dimer. Peptide sequencing demonstrated t his activity can be attributed to insulin degrading enzyme, a previous ly described member of the inverzincin family (Hooper, 1994). Kinetic studies with a number of peptide substrates indicate that the enzyme p referentially cleaves on the amino side of hydrophobic or basic residu es. However, the substrate specificity is more complex since not all b asic and hydrophobic residues in a peptide are cleaved. The enzyme exh ibits a requirement for a P'(2) residue. On the basis of k(cat)/K-m va lues, 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 k(cat), b ut a 10-fold decreased affinity relative to insulin. A role for insuli n-degrading enzyme as both a beta-endorphin-processing and -inactivati ng enzyme is implicated from these studies.