Aminopeptidases (EC.3.4.11) in alfalfa (Medicago sativa L.) leaves

Activities of two aminopeptidases and one iminopeptidase from mature alfalfa (Medicago sativa L.) leaf extracts were separated by acid precipitation, gel-filtration and ion-exchange chromatography. The two aminopeptidases AP1 and AP2, purified 70-fold and 130-fold respectively, showed pH optima of 7.5 with L-phenylalanine β-naphthylamide and 8.5 with L-alanine β-naphthylamide as substrates. Their activities were inhibited by cysteine protease inhibitors such as pCMB, pHMB and pHMPS. Iminopeptidase (IP) activity showed a pH optimum of 7.0 using L-proline β-naphthylamide as substrate. The iminopeptidase activity was greatly inhibited by pCMB, pHMB, pHMPS and NEM. Serine protease inhibitors did not affect IP activity. Molecular weights were estimated to be 70,000 and 93,000 for AP1 and AP2, respectively, using gel-filtration chromatography and SDS-PAGE. Abbreviations AP aminopeptidase; BANA N-a-benzoyl-DL-arginine-β-naphthylamide-hydrochloride; BAPNA N-a-benzoyl-DL-arginine-4-nitroanilid-hydrochloride; CBZ carbobenzoxy; DEAE diethylaminoethyl; EDTA ethylendiaminetetracetic acid; IP iminopeptidase; NA β-naphthylamide; NEM N-ethylmaleimide; pCMB p-chloromercuribenzoate; PMSF phenylmethylsulfonyl-fluoride; pHMB p-hydroxymercuribenzoate; pHMPS p-hydroxymercuriphenyl sulfonic acid; TLCK N-a-p-tosyl-L-lysine chloromethyl ketone; TPCK L-1-tosylamide-2-phenyl-ethylchloromethyl ketone