Isolation and characterization of okadaic acid binding proteins from the marine sponge Halichondria okadai.
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.
Sugiyama
Naoyuki
N
Department of Chemistry, School of Science, The University of Tokyo, Japan.
Konoki
Keiichi
K
Tachibana
Kazuo
K
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000378:metabolism*; D011161:Porifera / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D002352:Carrier Proteins; D019319:Okadaic Acid