Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence.
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.
Discovery Research Laboratories, Shionogi & Company, Fukushima-ku, Osaka 553-0002, Japan. koji.enomoto@shionogi.co.jp
Koji
K
Enomoto
Tohru
T
Nagasaki
Akira
A
Yamauchi
Junji
J
Onoda
Katsunori
K
Sakai
Tetsuya
T
Yoshida
Kazuhiko
K
Maekawa
Yuko
Y
Kinoshita
Ikuko
I
Nishino
Shino
S
Kikuoka
Takahiro
T
Fukunaga
Keiko
K
Kawamoto
Yoshito
Y
Numata
Hiroshi
H
Takemoto
Kiyoshi
K
Nagata
D000911:Antibodies, Monoclonal; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004791:Enzyme Inhibitors / Q000032:analysis; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D009942:Organometallic Compounds; D010798:Phycocyanin; D013050:Spectrometry, Fluorescence / Q000379:methods*; D011436:Spermidine Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology; D014230:Triazoles / Q000138:chemical synthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D009942:Organometallic Compounds; D014230:Triazoles; C002497:allophycocyanin; D010798:Phycocyanin; C070734:europium(III) trisbipyridine cryptate; C067418:5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione; D011436:Spermidine Synthase