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Journal of Biochemistry Advance Access originally published online on October 6, 2008
Journal of Biochemistry 2008 144(6):789-798; doi:10.1093/jb/mvn132
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Isolation and Biochemical Characterization of Two Forms of RD21 from Cotyledons of Daikon Radish (Raphanus sativus)

Yayoi Kikuchi, Hiroko Saika, Keizo Yuasa, Masami Nagahama and Akihiko Tsuji*

Department of Biological Science and Technology, The University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan

*To whom correspondence should be addressed. Tel: +81-88-656-7526, Fax: +81-88-655-3161, E-mail: tsuji{at}bio.tokushima-u.ac.jp

Received August 27, 2008; Accepted September 27, 2008


  Abstract

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.

Key Words: cleavage specificity, cysteine protease, germination, granulin domain, RD21

Abbreviations: ACTH, adrenocorticotropic hormone; Boc, t-butyloxycarbonyl; CA074, [N-(L-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-L-isoleucyl-L-proline]; E-64, trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane; Glt, glutaryl; MCA, 4-methylcoumaryl 7-amide; PMSF, phenylmethanesulfonyl fluoride; PVDF, polyvinylidene fluoride; pyr, L-pyroglutamyl; RD21, responsive to desiccation-21; TLCK, N{alpha}-p-tosyl-L-lysine chloromethyl ketone; Z, carbobenzoxy


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