E-Domain Peptide of Rat Proinsulin-Like Growth Factor-II: Validation of a Radioimmunoassay and Measurement in Culture Medium and Rat Serum*

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Abstract
We recently discovered a peptide derived from the carboxyl-terminal portion of the E-domain of rat proinsulin-like growth factor II (pro-IGF-II) in medium conditioned by BRL-3A rat liver cells. This peptide begins at residue 117 in the pro-IGF-II sequence. To measure physiological concentrations of this peptide in serum, We established an RIA for a synthetic peptide [rat pro-IGF-II-(117-156); E-domain peptide] corresponding to the carboxyl-terminal 40-amino acids of rat proIGF-II. The 41-residue peptide [Tyr116]pro-IGF-II-(117-156) was also synthesized and iodinated for use as tracer. Using polyclonal antibodies, we established a standard curve that measured as little as 25 pg/tube. Tracer was not displaced by insulin, human (h) IGF-I, hIGF-II, pro-hIGF-I-(71-105), rat GH, mouse EGF, ACTH, bovine PTH, ovine FSH, TRH, or LHRH under our assay conditions. However, a synthetic analog of the E-domain peptide [Phe117]pro-IGF-II-(118-156) showed displacement similar to that of the synthetic E-domain peptide. Serial dilutions of either culture medium or rat serum exhibited displacement parallel to the standard curve. Measurement of E-domain peptide in serum-free medium conditioned by BRL-3A rat liver cells showed a time-related increase in E-peptide concentration over a 72-h period. Analsysis of E-peptide immunoreactivity from the conditioned medium after gel filtration chromatography in 1 M acetic acid revealed a single peak which had a mol wt (determined by Western blot) identical to that of the synthetic E-peptide standard. The concentration of immunoreactive E-domain peptide levels in serum of 5-day-old rat pups was 30-40 times higher than concentrations in the serum of adult rats. Gel filtration chromatography of adult serum in 1 M acetic acid revealed a single major peak of immunoreactivity eluting at a position similar to the elution position of the E-domain peptide from BRL-3A rat liver cell-conditioned medium. The RIA described here should prove useful for measurement of the somatic output of E-domain peptide under different physiologicl conditions.

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