PurMcation and Some Properties of an of Cannabis sativa JSem Aminopeptidase frorn the Seeds

aminopeptidase [EC 3,4,11.1] from a plant source was rare.2'6) The cDNA sequence of a leucine aminopeptidase, which is an inducible component of the defense response in Io,copersicon esculentum, has been analyzed,'} and the enzymatic characteristics of this aminopeptidase have been studied.S} During screening for aminopeptidases from plant sources by using Leu:pNA as a substrate, we found a new aminopeptidase from hemp seeds. This paper describes the purification and some characterization ef this aminopeptidase (HSA). Hemp seeds (Ckennabis sativa) were obtained from Kagoshima City. Hemp seeds (2500g) were homogenized with a domestic mixer in 2500 ml of distilled water. The hornogenate was filtered through a cotton cloth and was centrifuged (4000 × g, for 30 min). Ammonium sulfate fractionation f the supernatant was done, and the 30-60% saturation-fraction was dialyzed and put on a DEAE-cellulose column (3.3 × 27 cm) equiiibrated with buffer A (50 mM Na, K-Pi buffer, pH 7.5). The enzyme was chromatographed with a linear gradient from buffer A (1 1) to buffer B (O.2 M Na, K-Pi buffer, pH 7.5, containing O.3 M NaCl, 1 1). The active fraction was put on a Sephadex G200 column (3.3 × 124cm) equilibrated with buffer A. The active fraction from the column was put on Sepabeads FP-OTi3 (Mitsubishi Chemicals, Tokyo) coiumn (1.8 × 36 cm) equilibrated with buffer C (50 mM Na, K-Pi buffer, pH 7.0, containing 30% saturated ammonium sulfate). The enzyme was eluted with a linear gradient from buffer C (500 ml) to buffer D (50 mM Na, K-Pi buffer, pH 7.0, 5ooml). The enzyme fraction was dialyzed against buffer E (20 mM Na, K-Pi buffer, pH 6.0) and put on a column of Leu-Sepharose (1.1 × 7.0