Evidence for the Existence of both Proteasomes and a Novel High Molecular Weight Peptidase in Entamoeba histolytica

• Published in: The Journal of biological chemistry 1996-03, Vol.271 (11), p.6212-6216
• Main Authors: Scholze, H, Frey, S, Cejka, Z, Bakker-Grunwald, T
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Centrifugation, Density Gradient; Coumarins - chemistry; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - isolation & purification; Cysteine Endopeptidases - metabolism; Entamoeba histolytica; Entamoeba histolytica - enzymology; Entamoeba histolytica - genetics; Fluorescent Dyes - chemistry; Kinetics; Microscopy, Electron; Molecular Sequence Data; Molecular Weight; Multienzyme Complexes - chemistry; Multienzyme Complexes - isolation & purification; Multienzyme Complexes - metabolism; Oligopeptides - chemistry; Peptide Fragments; Peptide Hydrolases - chemistry; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; Proteasome Endopeptidase Complex; Solubility; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.11.6212

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 of 385,000 ± 10%. On SDS gels, the purified enzyme exhibited a single band of M 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 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.