Isolation and biochemical characterization of two forms of RD21 from cotyledons of daikon radish (Raphanus sativus).
RD21 (Responsive to desiccation-21) is an Arabidopsis cysteine protease which possesses a granulin-like domain at the C-terminus. Although two forms of RD21 have been identified, consisting of an intermediate form (iRD21) containing a granulin domain and a mature form (mRD21) lacking this domain, the enzymatic properties of these enzymes remain poorly understood. In this study, mRD21 orthologue was purified to homogeneity from the cotyledons of daikon radish (Raphanus sativus). RD21 preferentially cleaved peptide bond that had an aromatic or hydrophobic amino acid at the P2 position. Furthermore, the presence of a polar amino acid at the P1 position enhanced the cleavage susceptibility of the peptide bond, although the importance of the type of amino acid residue at the P1 and P1' positions was not as significant as the residue located at the P2 position. The iRD21 was also identified as an oligomeric form by gel filtration and sedimentation analyses. The expression of RD21 mRNA was initiated by imbibition and continued at almost constant levels during germination. On the other hand, the enzyme activity increased markedly 5 days after imbibition. These results indicate that this elevation of RD21 activity is generated post-transcriptionally.
Kikuchi
Yayoi
Y
Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Saika
Hiroko
H
Yuasa
Keizo
K
Nagahama
Masami
M
Tsuji
Akihiko
A
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D018548:Cotyledon / Q000201:enzymology* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004399:Dynorphins / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D031224:Raphanus / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
D010940:Plant Proteins; D020033:Protein Isoforms; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D004399:Dynorphins; D003546:Cysteine Endopeptidases