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Schlagwörter:
Proteolysis; Purification; Complexes.; Biochemistry & biophysics.
Zusammenfassung:
To screen for high molecular weight proteases in Entamoeba histolytica, we subjected a soluble amebal extract to density gradient centrifugation and tested the fractions for activity against the chymotryptic peptide substrate, Suc-leucyl-leucyl-valyl-tyrosyl-4-methylcoumaryl-7-amide. Two peaks of activity, of approximately 11 and 20 S, were clearly separated. Based on SDS-electrophoretic pattern and immunoblot analysis, we ascribe the 20 S activity to proteasomes. The 11 S protein was purified from amebal homogenates by a series of chromatographic steps. As determined by molecular sieve chromatography and nondenaturing gel electrophoresis, the native complex had an apparent M(r) of 385,000 +/- 10%. On SDS gels, the purified enzyme exhib ited a single band of M(r) 62,000 that yielded a single N-terminal sequence, indicating that the preparation was homogeneous and that the native complex consisted of six very similar or identical subunits. The enzyme preferred peptides with aromatic residues at the P-1 position and had low but distinct activity toward azocasein. We conclude that the 11 S enzyme is a novel high molecular weight protease that is distinct from proteasomes. [References: 31]
