Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculetum, cv Bonnie Best) that had been spraye d with methyl jasmonate. For seedlings, cystatin purification was accomplis hed using EDTA washing, KCl extraction, 70 degrees C heat treatment, ammoni um sulfate fractionation and gel filtration chromatography. For mature plan ts, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cysta tin. Purified tomato cystatin has a molecular weight (M-r) of 88 kDa, eight papain binding domains, is a non-competitive inhibitor of papain with K-i of 1.4 nM and is not a glycoprotein. Tryptic peptides (M-r 26, 13 kDa) and most chymotryptic peptides (M-r 33, 13 kDa) of tomato cystatin retain inhib itor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser acco unted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4-11 at 4 degrees C, but unstable a t neutral pH at > 60 degrees C (Ea = 92.5 kJ/mole). Extracts of mature plan ts treated with methyl jasmonate contain lower M-r cystatins that appear to result from the action of an endogenous 26 kDa protease on the 88 kDa inhi bitor. (C) 2000 Elsevier Science Inc. All rights reserved.