Purification and characterization of two cysteine proteinases from Phaseolus vulgaris leaves

Abstract

The presence of cysteine, serine and aspartic proteinase activities in kidney bean ( Phaseolus vulgaris L.) leaves is shown. Two cysteine proteinases, named FLCP-1 and FLCP-3 ( Phaseolus vulgaris leaf cysteine proteinase 1 and 3), with molecular masses of 30 and 25.1 kDa, respectively, have been purified by gel filtration, two ion exchange chromatography, covalent chromatography on thiopropyl-Sepharose and again gel filtration. They are classified as cysteine proteinases on the ground of their inhibition by typical inhibitors and by their requirement of the presence of reducing agents. With isoelectric focusing, FLCP-1 gave two main bands with p1 4.85 and 4.45, while FLCP-3 focused at p1 4.5 and 4.3. Their specific activities towards benzoyloxycarbonyl-Phe-Arg-7(4-methyl)-coumarylamide (Z-Phe-Arg-MCA), Z-Arg-Arg-MCA and fluorescein thiocarbamoyl-haemoglobin (FTC-haemoglobin) were determined and FLCP-3 showed a higher activity for all three substrates. Both proteinases have alkaline pH optima for Z-Phe-Arg-MCA: FLCP-1 around pH 9.5 and FLCP-3 a broad optimum in the region of pH 7.5–9. The K i values for the inhibition of both proteinases with representatives of cystatins, stefins and Kunitz family cysteine proteinase inhibitors from potato were determined. All inhibited FLCP-1, with K i values ranging from 2 to 0.1 nM. The inhibition of FLCP-3 was even stronger with K i values from 0.5 nM to 40 pM. The inhibition of all enzyme-inhibitor pairs was competitive. Both proteinases were potently inhibited by an endogenous inhibitor from P. vulgaris seeds (FSCPI 5.5), which was detected immunologically in leaves. K i values were 0.15 nM and 30 pM for FLCP-1 and FLCP-3, respectively, indicating the possible physiological importance of this inhibitor.