Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus.
Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. However, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analysis, and the loop is shorter. In an effort to investigate the enzymatic properties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus) by chromatography through Sephacryl S-200, DEAE-cellulose, hydroxyapatite and organomercurial-Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS/PAGE under reducing conditions. The best synthetic substrate for CBCP was t-butyloxycarbonyl Leu-Arg-Arg-4-methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocorticotropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopeptidase activities. In addition, CBCP was found to display carboxymonopeptidase activity against the substrate o-aminobenzoyl-Phe-Arg-Phe(4-NO(2)). Daikon CBCP is less sensitive (1/7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination.
Tsuji
Akihiko
A
Department of Biological Science and Technology, University of Tokushima Graduate School, Japan. tsuji@bio.tokushima-u.ac.jp
Kikuchi
Yayoi
Y
Ogawa
Kentaro
K
Saika
Hiroko
H
Yuasa
Keizo
K
Nagahama
Masami
M
D002401:Cathepsin B; D002845:Chromatography; D018548:Cotyledon / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D018525:Germination; D006801:Humans; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D031224:Raphanus / Q000201:enzymology*; D013379:Substrate Specificity
D010940:Plant Proteins; D012333:RNA, Messenger; D003546:Cysteine Endopeptidases; D002401:Cathepsin B