Isolation and characterization of okadaic acid binding proteins from the marine sponge Halichondria okadai
Isolation and characterization of okadaic acid binding proteins from the marine sponge Halichondria okadai
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DOI：
10.1021/bi700490n
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发表时间：
2007-10-09
期刊：
影响因子：
2.9
通讯作者：
Tachibana, Kazuo
中科院分区：
文献类型：
--
作者：
Sugiyama, Naoyuki;Konoki, Keiichi;Tachibana, Kazuo
冈田酸（Okadaic acid）是一种蛋白磷酸酶PP 1和PP 2A的有效抑制剂，首次从海绵中分离得到。用生物素标记的光反应性冈田酸进行的光亲和标记实验表明，在H.冈代本文从H.通过[27-H-3]冈田酸的结合亲和力指导冈田。在SDS-PAGE分析中，OABP 1具有37 kDa的近似分子量。Edman降解后进行分子克隆和测序，鉴定OABP 1与兔PP 2A β催化亚基具有88%的同一性。另一方面，HPLC分析显示OABP 2由三种22 kDa蛋白质（OABP2.1、OABP2.2和OABP2.3）组成。电喷雾质谱分析表明OABP2.1和OABP2.2与冈田酸形成复合物。通过Edman降解和分子克隆确定的OABP 2的完整氨基酸序列显示，OABP 2.1与OABP 2.2具有96%的同一性，与OABP 2.3具有66%的同一性，而与迄今已知的任何蛋白磷酸酶具有非常轻微的同源性。OABP 2没有表现出磷酸酶活性，尽管它以0.97 nM的Kd与冈田酸结合。此外，OABP 2没有检测到海绵Halichondria japonica或甲藻Prorocentrum利马。因此，我们推测OABP 2可能参与冈田酸的解毒。
Okadaic acid, first isolated from the marine sponge Halichondria okadai, is a potent inhibitor of protein phosphatases 1and 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, ABP1 and OABP2 were purified from H. okadai as guided by the binding affinity of [27-H-3]okadaic acid. OABPl has an approximate molecular mass of 37 kDa in SDS-PAGE analysis. Edman degradation followed by molecular cloning and sequencing identified OABPl as being 88% identical to the rabbit PP2A beta 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 K-d 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.