Characterization of an aminopeptidase from grapes

The aminopeptidase activities from grapes, Vitis labruscana B. Takasumi, in creased parallel to the developmental stages and were, in order of activity , high for Phe-, Leu-, and Met-p-nitroanilides (aminoacyl-NAs). The activit ies were concentrated in the hypodermis from fully ripe grapes rather than in the flesh (mesocarp and placental tissue) or seeds. The rapid hydrolysis of the substrates was a common characteristic of the hypodermis of other s trains, indicating that Phe-specific aminopeptidase may play an important r ole in the hypodermis. An aminopeptidase was purified over 130-fold from th e hypodermis. The enzyme is a monomer of ca. 71-74 kDa as determined by SDS -PAGE and Sephacryl S-200 chromatography. The purified aminopeptidase, as w ell the crude extracts from whole grape and hypodermis, hydrolyzed aminoacy l-NAs with nonpolar side chains such as Phe and Leu more efficiently. The o rder of activity was similar to the crude extract of the hypodermis, indica ting that the enzyme may be a major aminopeptidase in the hypodermis. The e nzyme had a pH optimum of 7.0. The increase of activity up to pH 7.0 seems to be correlated with the increase in pH in the hypodermis during ripening. N-terminal-blocked Phe- and some oligopeptidyl-NAs as well as azocasein we re not hydrolyzed. Phenylmethylsulfonyl fluoride and N-ethylmaleimide inhib ited the enzyme in concentration- and time-dependent manners. To some exten t, aprotinin selectively decreased the activity. The enzyme was neither sig nificantly influenced by some metal ions nor inhibited by EDTA or 1,10-phen anthroline.