Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus
Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus
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DOI：
10.1093/jb/mvm119
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发表时间：
2007-08-01
影响因子：
2.7
通讯作者：
Honjo, Tsuneo
中科院分区：
文献类型：
--
作者：
Oba, Yumi;Shimasaki, Yohei;Honjo, Tsuneo
采用凝胶过滤层析、阴离子交换层析和聚丙烯酰胺凝胶电泳等方法，从腹腔注射三丁基锡（TBT）（5.0mg/kg体重）的牙鲆（Paralichthysolivaceus）血浆中分离纯化了TBT-bp 2蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明TBT-bp 2的分子量约为48 kDa，等电聚焦-聚丙烯酰胺凝胶电泳表明等电点约为3.0。TBT-bp 2含有40%的N-聚糖。通过快速扩增牙鲆肝组织cDNA末端和基因组步移技术，分别测定了TBT-bp 2的cDNA全序列和基因组序列。TBT-bp 2的216个氨基酸的序列与河豚鱼（Takifugu pardalis）石房蛤毒素和河豚毒素结合蛋白的序列有47%的同源性，而与TBT-bp 1的序列只有27%的同源性。对编码TBT-bp 2的氨基酸序列的基序序列和基因结构的分析表明，该蛋白属于脂质运载蛋白超家族。
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.