Related cystatin inhibitors from leaf and from seed Phaseolus vulgaris L.

The cysteine proteinase inhibitory activity in an extract of kidney bean (Phaseolus vulgaris L.) seed was purified using affinity chromatography on CM-papain Sepharose, and was resolved on gel filtration into three peaks, differing in their molecular masses. The third peak contained a major band of 14 kDa on SDS-PAGE and immunoblotting, and on isoelectric focusing showed an isoelectric point 5.5 and was therefore termed FSCPI 5.5 (P. vulgaris seed cysteine proteinase inhibitor). After additional purification on reverse phase HPLC N-terminal sequence of the inhibitor was determined. This shows FSCPI 5.5 to be a phytocystatin, whose N-terminus most closely resembles carrot cystatin with 48% identical amino acid residues, followed by leguminous phytocystatins, with which it shares 18-23% identical residues. FSCPI 5.5 strongly inhibited 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. Using a similar purification procedure a leaf inhibitor was purified to homogeneity. Its N-terminal amino acid sequence is similar but not identical to that of FSCPI 5.5. It is antigenically related to the seed inhibitor and has the same molecular mass. This inhibitor is the first member 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, respectively. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.