The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays.
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 3.4.22.34), 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.
Kembhavi
A A
AA
Department of Biochemistry, Strangeways Research Laboratory, Cambridge, United Kingdom.
Buttle
D J
DJ
Knight
C G
CG
Barrett
A J
AJ
D000595:Amino Acid Sequence; D015891:Cystatins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D010940:Plant Proteins; D010946:Plants, Medicinal; D011480:Protease Inhibitors / Q000494:pharmacology; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D000099240:Asparaginyl Endopeptidase
D015891:Cystatins; D003546:Cysteine Endopeptidases; D007460:Iodoacetamide; D007461:Iodoacetates; D019807:Iodoacetic Acid; D007930:Leucine; D010455:Peptides; D010940:Plant Proteins; D011480:Protease Inhibitors; D000099240:Asparaginyl Endopeptidase; C100158:cystatin, egg-white; C081576:vignain; C024974:E 64