J. Biochem, 1981, Vol. 89, No. 1 193-201
© 1981 Japanese Biochemical Society
research-article |
Purification and Properties of an Aminopeptidase from Seeds of Japanese Apricot
Department of Chemistry, Faculty of Science, Kyushu University 33 Higashi-ku, Fukuoka, Fukuoka 812
An aminopeptidase was purified about 1,700-fold from seeds of Japanese apricot (Prunus mume Sieb.) by a seven-step procedure comprising extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, first and second DEAE-Sepharose chromatography, hydroxyapatite chromatography, and Sephadex G-200 gel filtration. The purified enzyme was shown to be homogeneous on poly-acrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56,000 by gel filtration on Sephadex G-200 and the isoelectric point was 4.9. The pH optimum for L-leucine ß-naphthylamide was between pH 6.5 and 7.0, and the enzyme was stable in the pH 5.0 to 8.3 region and up to 50°C. The enzyme hydrolyzed a variety of aminopeptidase substrates with a free 
-amino group. Of the amino acid ß-naphthylamides, the enzyme was highly specific for substrates with a hydrophobic side chain in the amino terminal residue. The enzyme was strongly inhibited by p-chloromercuribenzoate, heavy metals, diethyl pyrocarbonate, and photooxidation with methylene blue, but was not affected by thiol compounds, peptidase inhibitors of microbial origin, such as bestatin and puromycin, or metal chelating agents. No activation of the enzyme by metal ions was observed. These results suggest that the enzyme is a true aminopeptidase in which cysteine and histidine residues participate in the catalytic process, and is not classifiable as a metalloenzyme.