Isolation and characterization of insulin-like growth factor-I from rainbow trout, Oncorhynchus mykiss.
Insulin-like growth factor-I (IGF-I) has been purified from plasma of adult rainbow trout, Oncorhynchus mykiss. Plasma samples were collected 48 hr following injection of recombinant tuna growth hormone at a dose of 0.5 microgram/g body weight. Acid-ethanol extract of plasma was fractionated by gel filtration on a Sephadex G-50 superfine column. Two-step ion-exchange chromatography on DEAE-52 and then Mono-S columns followed. Rainbow trout IGF-I was further purified by immunoaffinity chromatography with anti-recombinant coho salmon IGF-I (rsIGF-I) serum and HPLC on a reverse-phase C18 column. During purification, trout IGF-I was monitored by SDS-PAGE, immunoblotting with anti-IGF-I-serum, homologous radioimmunoassay (RIA) for salmon IGF-I, and sulfation bioassay. The trout IGF-I appeared on SDS-PAGE as a single band with a molecular weight of 7 kDa, the same size as rsIGF-I. The partial N-terminal amino acid sequence (residues 1-20) was identical to the predicted mature trout IGF-I cDNA sequence. Trout IGF-I cross-reacted with anti-rsIGF-I serum in immunoblotting and its dilution curve was parallel to the rsIGF-I standard curve in salmon RIA. In concentrations of 50 and 500 ng/ml, trout IGF-I significantly stimulated sulfation uptake by the cultured branchial cartilage of rainbow trout. This stimulatory effect of trout IGF-I was dose-dependent and similar in its biological potency to rsIGF-I.
School of Fisheries HF-15, University of Washington, Seattle 98195, USA.
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Moriyama
W W
WW
Dickhoff
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EM
Plisetskaya
D000595:Amino Acid Sequence; D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000097:blood*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000494:pharmacology
D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone