BIOSYNTHESIS INVITRO OF IMMUNOREACTIVE 31,000-DALTON CORTICOTROPIN-BETA-ENDORPHIN-LIKE MATERIAL BY BOVINE HYPOTHALAMUS

Enzymatically dispersed bovine hypothalamic or cortical tissue was maintained in culture in the presence of 3H-labeled amino acids. After such incubation, extracts of cells and of media contained 3H-labeled products that were specifically bound by immobilized affinity-purified antisera to corticotropin (ACTH) and β-endorphin. The majority of these products eluted in the void volume (Vo) upon Sephadex G-50 gel filtration; minor 3H-labeled products eluted in the regions of the ovine β-lipotropin marker and in fractions having apparent molecular weights of approximately 12,000 and 3800. Sequential use of these immobilized antisera revealed that most of the Vo material contained both ACTH and β-endorphin antigenic determinants within the same molecule(s), whereas retarded material contained only one of the determinants. When this Vo material was rerun on a Sephadex G-75 column, it coeluted with the 31-kilodalton precursor of both ACTH and β-endorphin obtained from a bovine anterior pituitary extract. Thus, the high molecular weight immunoreactive ACTH/β-endorphin-like 3H-labeled product(s) derived from the hypothalamic culture is similar to the pituitary-derived precursor in containing the dual antigenic determinants and in its gel filtration characteristics. In contrast, the cortex-derived cell preparation was devoid of 3H-labeled products specifically reactive with the antisera employed.