Characterization of Rat Hypothalamic Corticotropin-Releasing Factor by Reversed-Phase, High-Perf ormance Liquid Chromatography with Synthetic Ovine Corticotropin-Releasing Factor as a Marker

Abstract

Reversed-phase, high-performance liquid chromatography (HPLC) was carried out to characterize rat hypothalamic corticotropin-releasing factor (CRF) using synthetic ovine CRF as a marker. Samples were injected onto a stainless steel column (4 × 250 mm) packed with Hitachi gel 3053. The column was eluted using a gradient elution of increasing acetonitrile concentration, in a mixture of NaCl-HCl at a flow rate of 1.0 ml/min, monitoring the column effluent at 220 nm with an UV detector. Fractions eluted every 1–2 min were collected and lyophilized for subsequent CRF bioassay and radioimmunoassay. When various neuropeptide mixtures including synthetic ovine CRF were injected onto the column, synthetic ovine CRF was well separated from the other neuropeptides with a gradient of 0–60% acetonitrile in 0.1 M NaCl-0.01 N HCl or 0–80% acetonitrile in 0.05 M NaCl-0.01 N HCl. The median eminence extracts showed two main peaks of CRF bioactivity on HPLC. One (small CRF) coeluted with arginine vasopressin and oxytocin markers, and the other (big CRF) appeared near the position of synthetic CRF and was divided into two peaks. One coeluted with synthetic ovine CRF and the second eluted after synthetic CRF, showing high CRF activity. Three or four peaks of CRF immunoreactivity appeared on HPLC and the main peak appeared after synthetic ovine CRF marker. Our results suggest that rat CRF is different from ovine CRF, and the total lipophilicity of amino acid residues of rat CRF may be higher than that of ovine CRF.

Comparative Chromatography of Hypothalamic Corticotrophin-Releasing Factors

“…Many workers now agree on the multimolecular nature of CRF [9,19,20,23,27]. Several reports indicate that one of the hypothalamic CRFs has the apparent molecular size and CRF bioassay characteristics of VP [14,19,20,23,42,43] and this has been identified as AVP in the sheep [41] and lysine vasopressin (LVP) in the pig (this paper) by amino acid analysis.…”

“…Several reports indicate that one of the hypothalamic CRFs has the apparent molecular size and CRF bioassay characteristics of VP [14,19,20,23,42,43] and this has been identified as AVP in the sheep [41] and lysine vasopressin (LVP) in the pig (this paper) by amino acid analysis. However, the role that VP plays in ACTH re lease is still controversial [14,19,24,25,28,36,38].…”

Radioimmunoassay of CRF-Like Material in Rat Plasma: Validation of the Method

“…Since it is theoretically more de- expected to show more similarity to the native rat CRF than to the heptapeptide analog (20,21). In our preliminary report ( 13), we described the use of a Ty$'-CRF 36-41 synthetic analog of ovine CRF as labeled hormone, which gave standard curves parallel to those obtained when using the Tyro-CRF 1-41 analog.…”