利用均相时间分辨荧光开发高通量亚精胺合酶活性测定方法。

Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence.

作者信息

Enomoto Koji, Nagasaki Tohru, Yamauchi Akira, Onoda Junji, Sakai Katsunori, Yoshida Tetsuya, Maekawa Kazuhiko, Kinoshita Yuko, Nishino Ikuko, Kikuoka Shino, Fukunaga Takahiro, Kawamoto Keiko, Numata Yoshito, Takemoto Hiroshi, Nagata Kiyoshi

机构信息

Discovery Research Laboratories, Shionogi & Company, Fukushima-ku, Osaka 553-0002, Japan.

出版信息

Anal Biochem. 2006 Apr 15;351(2):229-40. doi: 10.1016/j.ab.2006.01.012. Epub 2006 Jan 26.

DOI:10.1016/j.ab.2006.01.012

PMID:16472757

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.

摘要

亚精胺合酶（SPDS）催化丙胺基团从脱羧S-腺苷甲硫氨酸（dcSAM）转移至腐胺，生成甲硫基腺苷（MTA）和亚精胺。SPDS在细胞增殖和分化中发挥调节作用。本文介绍了一种基于从作为供体的铕穴状化合物向作为受体的交联别藻蓝蛋白（XL665）的能量转移，使用均相时间分辨荧光（HTRF）进行高通量SPDS活性测定的方法。首先制备了一种高度特异性的抗MTA单克隆抗体MTA-7H8，然后使用铕穴状化合物标记的MTA-7H8和XL665标记的MTA开发了一种用于测定MTA的竞争性免疫测定法。在我们开发的均相免疫测定中，dcSAM与MTA-7H8的摩尔交叉反应百分比为0.01%，MTA的检测限为2.6 pmol/孔。我们的HTRF测定仅使用一个测定板，其中酶反应和MTA测定可以相继完成。因此，我们的方法能够从大量样品中自动筛选SPDS抑制剂。