A partial purification and characterization of two amino peptidases from Cucurbita maxima cotyledons

Abstract

An enzyme fraction containing two similar aminopeptidases has been isolated from Cucurbita maxima Duch. var. Hubbard cotyledons in purified form using l-leucine amide as the principal substrate. The substrate specificity of one of the two enzymes was similar to leucine aminopeptidase and the other varied only in that it hydrolyzed l-leucylglycylglycine at about three times the rate of leucine aminopeptidase. Evidence is presented that showed that a third peptidase which hydrolyzed l-leucylglycine more rapidly than either of the two aforementioned enzymes was also a constituent of squash cotyledons. This third peptidase was removed during the purification procedure and not studied in detail. The hydrolysis of l-leucine amide by the purified fraction was activated by Mg ++ and Mn ++ and inhibited by EDTA, (NH 4) 2SO 4 and high concentrations of NaCl. The highest pH optimum of the purified fraction was between 8·0 and 8·5, however there was also a lower pH optimum at pH 7·0. The peptidase activity of the fraction increased at a relatively uniform rate from 25 to 45°, increasing about three-fold within this range. The K m value was 2·4 × 10 −2 M/L with l-leucine amide as the substrate at pH 7·6.