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Journal of Biochemistry Advance Access originally published online on June 1, 2007
Journal of Biochemistry 2007 142(2):229-238; doi:10.1093/jb/mvm119
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© 2007 The Japanese Biochemical Society.

Purification and Characterization of Tributyltin-binding Protein Type 2 from Plasma of Japanese Flounder, Paralichthys olivaceus

Yumi Oba1, Yohei Shimasaki1,*, Yuji Oshima1, Hina Satone1, Takeshi Kitano2, Miki Nakao3, Shun-ichiro Kawabata4 and Tsuneo Honjo1

1Laboratory of Marine Environmental Science, Division of Marine Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan; 2Department of Materials and Life Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan; 3Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan; and 4Department of Biology, Faculty of Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan

*To whom correspondence should be addressed. Tel: +81-92-642-2906, Fax: +81-92-642-2908, E-mail: simasaki{at}agr.kyushu-u.ac.jp.

Received May 9, 2007; Accepted May 24, 2007


  Abstract

We used gel filtration chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis to purify tributyltin-binding protein type 2 (TBT-bp 2) from plasma of Japanese flounder (Paralichthys olivaceus) injected intraperitoneally with TBT (5.0 mg/kg body weight). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis indicated that the molecular mass of TBT-bp 2 was approximately 48 kDa, and isoelectric focusing–polyacrylamide gel electrophoresis indicated that the isoelectric point was approximately 3.0. TBT-bp 2 contained 40% N-glycan. The complete cDNA nucleotide sequence and the genome sequence of TBT-bp 2 were determined by means of rapid amplification of cDNA ends of liver tissue of Japanese flounder and a genome-walking technique, respectively. The 216 amino acid sequence of TBT-bp 2 showed 47% identity to the sequences of puffer fish (Takifugu pardalis) saxitoxin- and tetrodotoxin-binding protein but only 27% similarity to the sequence of TBT-bp 1. Analysis of the motif sequence of the amino acid sequence and the structure of the gene encoding TBT-bp 2 suggested that this protein belongs to the lipocalin superfamily.

Key Words: detoxification, glycoprotein, Japanese flounder, lipocalin superfamily, serum protein

Abbreviations: AGP, {alpha}1-acid glycoprotein; cds, coding sequences; dNTP, deoxyribonucleotide triphosphate; EST, expressed sequence tag; GSPs, gene-specific primers; GRE, glucocorticoid-response element; IEF–PAGE, isoelectric focusing–polyacrylamide gel electrophoresis; MT, metallothionein; native-PAGE, native-polyacrylamide gel electrophoresis; PFAs, perfluorinated alkylated substances; PFOS, perfluorooctanesulfonate; PSTBP, saxitoxin- and tetrodotoxin-binding proteins of the puffer fish (T. pardalis); RACE, rapid amplification of cDNA ends; TBT, tributyltin; TBT-bp, tributyltin-binding protein; TBTCl, tributyltin chloride; TPhTCl, triphenyltin chloride

Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB277758 and AB277759.


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