ISOLATION AND CHARACTERIZATION OF INSULIN-LIKE GROWTH-FACTOR-I FROM RAINBOW-TROUT, ONCORHYNCHUS-MYKISS

Insulin-like growth factor-I (IGF-T) has been purified from plasma of adult rainbow trout, Oncorhynchns mykiss. Plasma samples were collected 48 hr following injection of recombinant tuna growth hormone at a dose of 0.5 mu g/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 TGF-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. (C) 1995 Academic Press,Inc.