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

Abstract

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculentum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCl extraction, 70°C heat treatment, ammonium sulfate fractionation and gel filtration chromatography. For mature plants, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cystatin. 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 inhibitor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser accounted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4–11 at 4°C, but unstable at neutral pH at >60°C (Ea=92.5 kJ/mole). Extracts of mature plants 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 inhibitor.