| Literature DB >> 16472757 |
Koji Enomoto1, Tohru Nagasaki, Akira Yamauchi, Junji Onoda, Katsunori Sakai, Tetsuya Yoshida, Kazuhiko Maekawa, Yuko Kinoshita, Ikuko Nishino, Shino Kikuoka, Takahiro Fukunaga, Keiko Kawamoto, Yoshito Numata, Hiroshi Takemoto, Kiyoshi Nagata.
Abstract
Spermidine synthase (SPDS) catalyzes transfer of the propylamine group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine to yield methylthioadenosine (MTA) and spermidine. SPDS plays a regulatory role in cell proliferation and differentiation. This article describes the development of a high-throughput SPDS activity assay using homogeneous time-resolved fluorescence (HTRF) based on energy transfer from europium cryptate as a donor to crosslinked allophycocyanin (XL665) as an acceptor. First a highly specific anti-MTA monoclonal antibody, MTA-7H8, was generated, and then a competitive immunoassay for MTA determination was developed using europium cryptate-labeled MTA-7H8 and XL665-labeled MTA. In our homogeneous immunoassay, the percentage molar cross-reactivity of dcSAM with MTA-7H8 was 0.01% and the detection limit of MTA was 2.6 pmol/well. Our HTRF assay uses only one assay plate in which both enzyme reaction and MTA determination can be done successively. Therefore, our method can enable automatic screening of SPDS inhibitors from large numbers of samples.Entities:
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Year: 2006 PMID： 16472757 DOI： 10.1016/j.ab.2006.01.012
Source DB: PubMed Journal: Anal Biochem ISSN： 0003-2697 Impact factor: 3.365