RELATED CYSTATIN INHIBITORS FROM LEAF AND FROM SEED PHASEOLUS-VULGARIS L

The cysteine proteinase inhibitory activity in an extract of kidney be an (Phaseolus vulgaris L.) seed was purified using affinity chromatogr aphy on CM-papain Sepharose, and was resolved on gel filtration into t hree peaks, differing in their molecular masses. The third peak contai ned a major band of 14 kDa on SDS-PAGE and immunoblotting, and on isoe lectric focusing showed an isoelectric point 5.5 and was therefore ter med FSCPI 5.5 (P. vulgaris seed cysteine proteinase inhibitor). After additional purification on reverse phase HPLC N-terminal sequence of t he inhibitor was determined. This shows FSCPI 5.5 to be a phytocystati n, whose N-terminus most closely resembles carrot cystatin with 48% id entical amino acid residues, followed by leguminous phytocystatins, wi th which it shares 18-23% identical residues. FSCPI 5.5 strongly inhib ited two P. vulgaris leaf cysteine proteinases, FLCP-1 and FLCP-3 and was found to be a potent inhibitor of papain and human cathepsins B, H and L, with K-i values 0.08, 3.6, 2.8 and 0.02 nM, respectively. Usin g a similar purification procedure a leaf inhibitor was purified to ho mogeneity. Its N-terminal amino acid sequence is similar but not ident ical to that of FSCPI 5.5. It is antigenically related to the seed inh ibitor and has the same molecular mass. This inhibitor is the first me mber of the cystatin superfamily to be characterized from plant leaves . It inhibited papain with K-i value of 0.18 nM, and FLCP-1 and FLCP-3 , its potential target enzymes, with K-i values of 0.06 and 0.04 nM, r espectively. (C) 1998 Elsevier Science Ireland Ltd. All rights reserve d.