A novel endopeptidase with a strict specificity for threonine residues at the P1 ' position

An endopeptidase was purified from Archachatina ventricosa by chromatograph y on columns of gel filtration, DEAE-Sepharose and phenyl-Sepharose, The pr eparation was shown to be homogeneous by polyacrylamide gel electrophoresis and capillary electrophoresis. The purified enzyme displayed two protein b ands on SDS-polyacrylamide gel electrophoresis with estimated molecular wei ghts of 90,000 and 121,000. The protease exhibited maximum proteolytic acti vity at 55 degrees C and at pH 8.0, but it retained more than 85% of its ac tivity in the pH range 7.5 to 8.5. It was completely inactivated by the che lating agents EDTA and 1,10-phenanthroline which are metalloprotease inhibi tors. Studies on substrate specificity showed that only the amide bonds of peptide substrates having a threonine residue at the P1' position were hydr olyzed by the purified protease, This endopeptidase constitutes a novel too l for the study of proteins in view of its narrow and unique substrate spec ificity. (C) 1999 Academic Press.