title: Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination.
abstract: Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.
authors.affiliation: Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjima, Tokushima 770-8506, Japan. tsuji@bio.tokushima-u.ac.jp
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tsuji
authors.forename: Kana
authors.initials: K
authors.lastname: Tsukamoto
authors.forename: Keiko
authors.initials: K
authors.lastname: Iwamoto
authors.forename: Yuka
authors.initials: Y
authors.lastname: Ito
authors.forename: Keizo
authors.initials: K
authors.lastname: Yuasa
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018548:Cotyledon / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018525:Germination; D007202:Indicators and Reagents / Q000378:metabolism; D007564:Japan; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D031224:Raphanus / Q000201:enzymology* / Q000254:growth & development* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D004151:Dipeptides; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; D003553:Cystine; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D003546:Cysteine Endopeptidases; D003545:Cysteine