Biological and immunological studies of bovine hypothalamic corticotropin-releasing factor

Corticotropin-releasing factor B (CRF-B) is a peptide(s) isolated from bovine hypothalamic extracts by Sephadex G-100 chromatography on the basis of its ability to stimulate secretion of adrenocorticotropin (ACTH) in vitro and in vivo. It is similar in molecular size to the 41-residue ovine CRF (oCRF) or rat CRF (rCRF) recently elucidated and appears to be their bovine counterpart. Immunoreactivity of CRF-B was examined in homologous radioimmunoassays (RIAs) for oCRF or rCRF, using several anti-oCRF and anti-rCRF antibodies. CRF-B cross-reacted well with anti-oCRF antibodies but poorly with anti-rCRF antibodies. Purification of CRF-B with preparative isoelectric focusing yielded four CRF peaks, B-1 (pH 4.7), B-2 (pH 5.5), B-3 (pH 6.3), and B-4 (pH 7.0), which accounted for 16, 30, 46, and 8% of the total immunoreactivity, respectively. CRF B-2, B-3, and B-4 showed both immunological activity and biological activity in vitro (cell culture assay) and in vivo (Arimura assay), whereas CRF B-1 showed only immunoreactivity. Their relative bioactivity/immunoreactivity ratios were 0 (B-1), 1 (B-2), 1 (B-3), and 3 (B-4). All of these CRF-B subtypes exhibited RIA displacement curves parallel to that for the oCRF standard and coeluted with oCRF on Sephadex G-100 chromatography, which suggests that their molecular modifications are relatively minor.