STABILITY AND OLIGOMERIC EQUILIBRIA OF REFOLDED INTERLEUKIN-1-BETA CONVERTING-ENZYME

We report the preparation and characterization of interleukin-1 beta c onverting enzyme (ICE) refolded from its p20 and p10 protein fragments , Refolded ICE heterodimer (p20p10) was catalytically active but unsta ble, and in size exclusion chromatography eluted at an apparent molecu lar mass of 30 kDa, The mechanisms of the observed instability were pH -dependent dissociation at low enzyme concentrations, and autolytic de gradation of the p10 subunit at high concentrations, Binding and subse quent removal of a high affinity peptidic inhibitor increased the appa rent molecular mass to 43 kDa (by size exclusion chromatography), and significantly increased its stability and specific activity. Chemical cross-linking and SDS-polyacrylamide gel electrophoresis analysis of t he 43-kDa size exclusion chromatography conformer revealed a 60-kDa sp ecies, which was absent in the 30-kDa conformer, suggesting that inhib itor binding caused formation of a (p20p10), homodimer, The observatio n of a reversible equilibrium between ICE (p20p10) and (p20p10)(2) sug gests that analogous associations, possibly between ICE and ICE homolo gs, can occur in vivo, resulting in novel oligomeric protease species.