S. Moriyama et al., ISOLATION AND CHARACTERIZATION OF INSULIN-LIKE GROWTH-FACTOR-I FROM RAINBOW-TROUT, ONCORHYNCHUS-MYKISS, General and comparative endocrinology, 99(2), 1995, pp. 221-229
Insulin-like growth factor-I (IGF-T) has been purified from plasma of
adult rainbow trout, Oncorhynchns mykiss. Plasma samples were collecte
d 48 hr following injection of recombinant tuna growth hormone at a do
se of 0.5 mu g/g body weight. Acid-ethanol extract of plasma was fract
ionated by gel filtration on a Sephadex G-50 superfine column. Two-ste
p ion-exchange chromatography on DEAE-52 and then Mono-S columns follo
wed. Rainbow trout IGF-I was further purified by immunoaffinity chroma
tography with anti-recombinant coho salmon IGF-I (rsIGF-I) serum and H
PLC on a reverse-phase C18 column. During purification, trout IGF-I wa
s monitored by SDS-PAGE, immunoblotting with anti-IGF-I-serum, homolog
ous radioimmunoassay (RIA) for salmon IGF-I, and sulfation bioassay. T
he 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 amin
o 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 rs
IGF-I standard curve in salmon RIA. In concentrations of 50 and 500 ng
/ml, trout IGF-I significantly stimulated sulfation uptake by the cult
ured branchial cartilage of rainbow trout. This stimulatory effect of
trout IGF-I was dose-dependent and similar in its biological potency t
o rsIGF-I. (C) 1995 Academic Press,Inc.