Insulin-like growth factor-I and insulin-like growth factor-binding protein in the toad, Bufo woodhousei

Abstract

Molecular weight characteristics and plasma concentrations of insulin-like growth factor-I (IGF-I) and its binding protein (IGF-BP) were investigated in the toad, Bufo woodhousei. IGF-I and IGF-BP were measured by radioimmunoassay (RIA, K d = 0.37 ± 0.04 ng/ml) and charcoal-separated ligand binding assay, respectively, in male toad plasma and adult male human donor plasma using a synthetic human IGF-I standard. Prior to the IGF-I RIA, samples were acid-ethanol extracted. Molecular weight characteristics were determined using size exclusion chromatography. At neutral pH (pH = 7.4), IGF-I immunoreactivity and IGF-BP eluted at molecular weight >66 kDa in both toad and human plasma. Acid chromatography (pH ∼3) resulted in the separation of IGF-I from its binding protein and consequently a shift of IGF-I immunoreactivity to the low molecular weight fractions (∼8 kDa) for both toad and human. IGF-BP activity shifted to molecular weight ∼50 kDa. Toad plasma IGF-I and IGF-BP activity exhibited differences according to season: IGF-I levels were low in the spring (March = 0.48 ± 0.11 ng eq/ml), increased progressively to reach a peak in July (5.84 ± 2.5 ng eq/ml), and decreased to low levels again in the fall (October = 0.60 ± 0.08, November = 0.45 ± 0.09 ng eq/ml). Plasma IGF-BP activity demonstrated a similar pattern (March = 17.4 ± 2.5, July = 35.0 ± 2.4, November = 12.6 ± 3.2% specific binding). IGF-I was produced for at least 72 hr when toad liver explants were cultured in serum-free medium, indicating that the liver is a source of IGF-I in anurans.