Purification and identification of a tributyltin-binding protein from serum of Japanese flounder, Paralichthys olivaceus.
Tributyltin (TBT) is an industrial chemical used as an antifoulant in marine environments. Previously, we reported that TBT accumulates in the serum or plasma of some fishes and is bound to a high molecular weight compound in the serum of the Japanese flounder, Paralichthys olivaceus. In this study, we succeeded in purifying the TBT-binding protein (TBT-bp) from the serum of Japanese flounder by using gel filtration chromatography, anion exchange chromatography, and polyacrylamide gel electrophoresis, with a 2.6% yield and a 77-fold purification. The molecular mass of TBT-bp was approximately 46.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was approximately 3.0 on isoelectric focusing-polyacrylamide gel electrophoresis. The TBT-bp contained 42% N-glycan. The cDNA nucleotide sequence of TBT-bp was determined by reverse transcription-polymerase chain reaction of Japanese flounder liver, and we deduced a sequence of 191 amino acids of mature TBT-bp. No sequence identical to the TBT-bp amino acid sequence was found within the SWISS-PROT (http://www.nig. ac.jp/) protein database; however, a lipocalin-like sequence pattern was observed. We concluded that the TBT-bp was a novel protein that has not yet been reported, although some DNA sequences from expressed sequence tags (ESTs) of Japanese flounder liver had a high identity. A high expression level of TBT-bp gene was found in the liver, but the gene was slightly detectable in the kidney and brain.
Shimasaki
Yohei
Y
Laboratory of Marine Biochemistry, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
Oshima
Yuji
Y
Yokota
Yoshiko
Y
Kitano
Takeshi
T
Nakao
Miki
M
Kawabata
Shun-ichiro
S
Imada
Nobuyoshi
N
Honjo
Tsuneo
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005432:Flounder / Q000502:physiology*; D005786:Gene Expression Regulation; D008099:Liver / Q000502:physiology; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014220:Trialkyltin Compounds / Q000378:metabolism*
D002352:Carrier Proteins; D014220:Trialkyltin Compounds; C011559:tributyltin