Posttranslational processing of corticotropin-releasing factor in the ovine tuberoinfundibular system and pituitary

The present studies were undertaken to characterize the immunoreactive-corticotropin-releasing factor (ir-CRF) in two areas of the ovine tuberoinfundibular system, hypophysial portal blood, and pituitary. With an antiserum raised against synthetic ovine (o)CRF(1-41) and 125I-Tyro-oCRF(1-41) as the tracer, concentrations of ir-CRF (pg/mg wet weight, n = 5) were: paraventricular hypothalamus (PVN), 11.7 +/- 2.5; median eminence (ME), 2276 +/- 296; anterior pituitary (AP), less than 0.5; posterior pituitary (PP), 10.0 +/- 2.2. Analysis of the ir-CRF in these areas on G-75 Sephadex chromatography revealed two main peaks--a 'major' peak which coeluted with synthetic oCRF(1-41) and a 'minor' peak which eluted eight fractions later. These two immunoreactive species of CRF were also found in hypophysial portal blood. When ME extract was analyzed by reverse-phase high performance liquid chromatography (HPLC), the 'minor' peak of ir-CRF eluted before that of CRF(1-41). Since CRF contains Arg35-Lys36 within its sequence, we tested the hypothesis that the 'minor' peak of ir-CRF represented a fragment, or fragments, of the molecule derived by proteolytic cleavage at this site. Tyro-oCRF(34-41) was digested with trypsin and the reaction products were identified by amino acid analysis. Two of these products were CRF(36-41) and CRF(37-41), and both migrated in the 'minor' peak area on G-75 Sephadex chromatography and HPLC. In the CRF(1-41) RIA, serial dilution of both fragments yielded nonparallel displacement curves. However, with 125I-Tyro-oCRF(34-41) as the radiolabeled ligand and Tyro-oCRF(34-41) as the standard, serial dilutions of CRF(1-41), CRF(36-41), and CRF(37-41) generated parallel displacement curves, and the molar cross-reactivities were 90%, 45% and 10% respectively. When the ir-CRF in HPLC fractions of ovine ME was measured in the Tyro-oCRF(34-41) RIA, the molar abundance of the hexapeptide and pentapeptide could be obtained. Calculations based on the premise that the 'minor' peak was solely composed of either the hexapeptide or pentapeptide indicated that CRF(36-41) could account for up to 37% of the total ir-CRF, or that CRF(37-41) could account for up to 73% of the total immunoreactivity. On more discriminating HPLC systems, immunoreactive (ir-) oCRF in the sheep median eminence (ME) could be resolved into five different molecular forms. On three distinct chromatographic systems, four of these immunoreactive species shared retention characteristics identical with synthetic oCRF(37-41), oCRF(36-41), oCRF(16-41) and oCRF(1-41), with the fifth immunoreactive peak as yet unidentified.(ABSTRACT TRUNCATED AT 400 WORDS)