Molecular characterization of phytocystatins isolated from Chinese cabbage flower buds

Chinese cabbage cDNA clones (BCPI-1, -2, and -3) encoding phytocystatin were characterized. The deduced BCPI amino acid sequences contained the consensus motifs that have been shown to interact with the active site of cysteine peptidases (CysPs). BCPI-1 and -2, but not BCPI-3, contained an extended carboxyl-terminal region that included a cysteine residue. BCPI-1 and -2 existed both as monomers and dimers. The monomeric forms of BCPI-1 (K-i = 6.84 +/- 0.3 x 10(-8) M) and BCPI-2 (K-i = 6.77 +/- 0.2 x 10(-8) M) inhibited papain equimolar complexes in competition with their substrates. The inhibitory activity was clearly reduced in the pH range of 7.0-11.5. In contrast, BCPI-3 was present only as a 16 kDa monomer, and had a K-i value of 6.14 +/- 4 x 10(-8) M against papain. It was highly stable over wide ranges of pH values and temperature. The differences between the BCPIs with respect to protein stability and inhibitory activity suggest that they may play diverse physiological roles in Chinese cabbage, and may interact with cysteine peptidases through different conditions.