E-domain peptide of rat proinsulin-like growth factor-II: validation of a radioimmunoassay and measurement in culture medium and rat serum.
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 pro-IGF-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. Analysis of E-peptide immunoreactivity from 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 rat 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 physiological conditions.
D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D002850:Chromatography, Gel; D003470:Culture Media; D007106:Immune Sera / Q000276:immunology; D007335:Insulin-Like Growth Factor II / Q000032:analysis* / Q000097:blood / Q000276:immunology; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D010446:Peptide Fragments / Q000032:analysis* / Q000097:blood / Q000276:immunology; D011498:Protein Precursors; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013002:Somatomedins / Q000032:analysis*
D003470:Culture Media; D007106:Immune Sera; D010446:Peptide Fragments; D011498:Protein Precursors; D013002:Somatomedins; C052252:proinsulin-like growth factor II (117-156); D007335:Insulin-Like Growth Factor II