The E-domain peptide of rat pro-insulin-like growth factor II (proIGF-II): Properties of the peptide in serum and production by rat cell lines

Abstract

We previously identified a naturally occurring peptide fragment derived from the carboxyl terminal region of the E-domain of pro-insulin-like growth factor II (proIGF-II 117–156) in medium conditioned by cultured BRL-3A rat liver cells. In the present study we utilized a radioimmunoassay (RIA) for this peptide to measure physiological concentrations of the peptide in media and serum. Serum levels of the E-domain peptide were very high in the 5-day neonatal rat and declined thereafter to reach low levels in adult rat serum. Chromatography of adult rat serum on Sephadex G-75 in 1 M acetic acid yielded a single broad peak of E-peptide immunoreactivity that coeluted with a synthetic E-peptide standard. However, chromatography of day 5 neonatal rat serum on Sephadex G-75 yielded two peaks of immunoreactivity. One of the peaks coeluted with a synthetic E-peptide standard, whereas the other peak eluted in a region where higher molecular weight proteins typically elute. Experiments aimed at determining whether adult rat serum contained a binding protein for the E-domain peptide revealed that: (1) serum contains little, if any, binding protein for the E-domain peptide, (2) serum contains a proteinase activity that degrades the E-domain peptide, and (3) the proteinase activity can be eliminated by acetic acid/ethanol extraction. Of several rat cell lines tested (BRL-3A, rat embryo fibroblasts (REF), hepatoma cell lines (H4, HTC), GH 3 pituitary tumor cells, and normal rat kidney fibroblasts (NRK)), only BRL-3A and REF cells secreted measurable E-domain peptide into the medium. In addition, it was found that some component(s) of serum could stimulate secretion of E-domain peptide from BRL-3A and REF cells. Chromatography of the immunoreactivity from BRL-3A and REF-conditioned media on Sephadex G-75 in 1 M acetic acid yielded a single peak that coeluted with a synthetic E-domain peptide standard. Since secretion of the E-domain peptide parallels the expression of IGF-II, the RIA for the proIGF-II E-domain peptide may be useful for studies of the biosynthesis and secretion of IGF-II under different physiological conditions. The RIA for the IGF-II E-domain peptide has two technical advantages over the RIA for IGF-II, namely, the lack of interference by IGF binding proteins and the relative ease with which large quantities of pure antigen can be synthesized.