Abstract
A cationic peptidase, named hemisphaericin-C, has been purified from the juice of Bromelia hemisphaerica fruits by ammonium sulfate precipitation, gel filtration on Sephadex G-75 and cationic exchange chromatography on carboxymethyl cellulose (CMC), to yield a single 24 kDa band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which showed esterase and proteolytic activities. The esterase activity was inhibited by E-64, leupeptin, and cystatin, but not by EDTA. Characterization of the primary specificity of hemisphaericin-C showed activity towards substrates specific for chymotrypsin: N-succinyl-L-Phe-p-nitroanilide (PHEpNA) and N-benzoyl-L-Tyr-p-nitroanilide (TYRpNA), and those for trypsin: N-benzoyl-L-arg-p-nitroanilide (BApNA) to a lower degree. The higher selectivity, assessed by Vmax/Km, was obtained for PHEpNA, the substrate containing the aromatic lateral chain amino acid at the P1 position. The preference of hemisphaericin-C for PHEpNA gives a clue in the search for a chymotrypsin-like peptidase from a vegetal source.