Purification of Hypothalamic Corticotrophin-Releasing Factor (CRF) Of Ovine Origin

Abstract

Summary Acetone powders were prepared from large numbers of ovine SME fragments. After preliminary extraction of the powder with glacial acetic acid and lyophilization, the resultant powder was extracted with 0.1 M ammonium acetate and subjected to gel filtration on a long column of Sephadex G-25. A wide zone was eluted from the column which induced adrenal ascorbic acid depletion upon its iv administration into rats with median eminence lesions. Part of this activity was caused by ACTH-like substances, since it was still present in hypophysectomized rats; however, this active zone was free of pressor activity. The active zones from several fractionations with Sephadex were pooled and placed on a CMC column. Elution was accomplished by application of gradients of ammonium acetate buffer of increasing ionic strength and pH. A narrow zone was eluted from the column which was active in inducing adrenal ascorbic acid depletion in rats with lesions, but was inactive in hypophysectomized rats. A dose of < 3 μg of peptide had significant activity. This method appears applicable for preparation of highly purified hypothalamic CRF. In this study we were able to locate only one CRF; however, the possible existence of other CRF's is not excluded.