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Stability and oligomeric equilibria of refolded interleukin-1beta converting enzyme
R V Talanian1, L C Dang, C R Ferenz
1BASF Bioresearch Corporation, Worcester, Massachusetts 01605, USA.
The Journal of Biological Chemistry
|September 6, 1996
Summary
We refolded interleukin-1beta converting enzyme (ICE) from protein fragments. Inhibitor binding stabilized ICE and induced homodimer formation, suggesting in vivo oligomerization of ICE and related proteases.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Interleukin-1beta converting enzyme (ICE) is a key protease.
- Understanding ICE's structure-function relationship is crucial for its inhibition and therapeutic targeting.
Purpose of the Study:
- To characterize refolded ICE from its constituent protein fragments.
- To investigate the effects of inhibitor binding on ICE stability, activity, and oligomeric state.
Main Methods:
- Protein refolding from p20 and p10 fragments.
- Size exclusion chromatography to assess molecular mass and stability.
- Chemical cross-linking and SDS-PAGE to determine oligomeric state.
Main Results:
- Refolded ICE (p20p10) was active but unstable, dissociating at low concentrations and degrading at high concentrations.
- High-affinity inhibitor binding increased ICE's apparent molecular mass to 43 kDa, enhancing stability and specific activity.
- Inhibitor binding induced formation of a (p20p10)2 homodimer, evidenced by a shift to a 60-kDa species.
Conclusions:
- ICE exists in a reversible equilibrium between monomeric (p20p10) and dimeric (p20p10)2 forms.
- Inhibitor binding promotes homodimerization, suggesting potential in vivo oligomerization of ICE and homologous proteases.
- This oligomerization may lead to novel protease species with distinct functions.