Sugiyama Naoyuki, Konoki Keiichi, Tachibana Kazuo
Department of Chemistry, School of Science, The University of Tokyo, Japan.
Biochemistry. 2007 Oct 9;46(40):11410-20. doi: 10.1021/bi700490n. Epub 2007 Sep 15.
Okadaic acid, first isolated from the marine sponge Halichondria okadai, is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A, respectively). Photoaffinity labeling experiments previously performed with biotinylated photoreactive okadaic acid revealed the presence of okadaic acid binding protein (OABP) in the crude extract of H. okadai. In this article, OABP1 and OABP2 were purified from H. okadai as guided by the binding affinity of [27-3H]okadaic acid. OABP1 has an approximate molecular mass of 37 kDa in SDS-PAGE analysis. Edman degradation followed by molecular cloning and sequencing identified OABP1 as being 88% identical to the rabbit PP2Abeta catalytic subunit. On the other hand, HPLC analysis revealed that OABP2 consists of three 22 kDa proteins (OABP2.1, OABP2.2, and OABP2.3). Electrospray ionization mass spectrometry indicated that OABP2.1 and OABP2.2 form a complex with okadaic acid. The complete amino acid sequence of OABP2, determined by Edman degradation and molecular cloning, showed that OABP2.1 is 96% identical to OABP2.2 and 66% identical to OABP2.3, while being very slightly homologous to any protein phosphatases known to date. OABP2 did not exhibit phosphatase activity, though it bound to okadaic acid with a Kd of 0.97 nM. Furthermore, OABP2 was not detected in the sponge Halichondria japonica or the dinoflagellate Prorocentrum lima. We thus speculated that OABP2 might be involved in detoxifying okadaic acid.
冈田酸最初是从海洋海绵冈村氏软海绵中分离出来的，是一种强效的蛋白磷酸酶1和2A（分别为PP1和PP2A）抑制剂。先前用生物素化的光反应性冈田酸进行的光亲和标记实验表明，冈村氏软海绵的粗提物中存在冈田酸结合蛋白（OABP）。在本文中，根据[27-³H]冈田酸的结合亲和力，从冈村氏软海绵中纯化出了OABP1和OABP2。在SDS-PAGE分析中，OABP1的近似分子量为37 kDa。通过埃德曼降解法，随后进行分子克隆和测序，确定OABP1与兔PP2Abeta催化亚基的同源性为88%。另一方面，HPLC分析表明，OABP2由三种22 kDa的蛋白质（OABP2.1、OABP2.2和OABP2.3）组成。电喷雾电离质谱表明，OABP2.1和OABP2.2与冈田酸形成复合物。通过埃德曼降解法和分子克隆确定的OABP2的完整氨基酸序列表明，OABP2.1与OABP2.2的同源性为96%，与OABP2.3的同源性为66%，而与迄今为止已知的任何蛋白磷酸酶的同源性都非常低。OABP2虽然以0.97 nM的解离常数与冈田酸结合，但不表现出磷酸酶活性。此外，在海绵日本软海绵或鞭毛藻利马原甲藻中未检测到OABP2。因此，我们推测OABP2可能参与了对冈田酸的解毒作用。
