The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays

Arch Biochem Biophys. 1993 Jun;303(2):208-13. doi: 10.1006/abbi.1993.1274.


Two endopeptidases are present in the seeds of Vigna aconitifolia (moth bean), and their activities increase during germination. One enzyme, which we term "vignain," can be assayed with benzyloxycarbonyl-phenylalanyl-arginyl-7-(4-methyl)coumarylamide as substrate. The second is legumain (EC, which can be assayed with benzyloxycarbonyl-alanyl-alanyl-asparaginyl-7-(4-methyl)-coumarylamide. The enzymes were purified, and their specificities for substrates and inhibitors were examined. Vignain has properties expected of a cysteine endopeptidase of the papain family, with the exception of a remarkably low reactivity with iodoacetate. Legumain is a very atypical cysteine endopeptidase, being insensitive to inhibition by chicken cystatin and E-64 (L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4-guanidino )butane), and reacting more rapidly with iodoacetamide than with iodoacetate. We discuss our findings in relation to the literature on the proteolytic enzymes of legume seeds.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Cystatins / pharmacology
  • Cysteine Endopeptidases / isolation & purification*
  • Cysteine Endopeptidases / metabolism
  • Enzyme Stability
  • Fabaceae / enzymology*
  • Hydrogen-Ion Concentration
  • Iodoacetamide / pharmacology
  • Iodoacetates / pharmacology
  • Iodoacetic Acid
  • Leucine / analogs & derivatives
  • Leucine / pharmacology
  • Molecular Sequence Data
  • Molecular Weight
  • Peptides / metabolism
  • Plant Proteins*
  • Plants, Medicinal*
  • Protease Inhibitors / pharmacology
  • Seeds / enzymology*
  • Substrate Specificity


  • Cystatins
  • Iodoacetates
  • Peptides
  • Plant Proteins
  • Protease Inhibitors
  • cystatin, egg-white
  • Cysteine Endopeptidases
  • vignain
  • asparaginylendopeptidase
  • Leucine
  • E 64
  • Iodoacetic Acid
  • Iodoacetamide