Analysis of the common precursor to corticotropin and endorphin.

Cells of the AtT-20/D-16v mouse pituitary tumor cell line synthesize corticotropin (ACTH) and endorphin as part of the same common precursor molecule (Mains, R. E., and Eipper, B. A. (1978) J. Biol. Chem. 253, 651-655). The entire structure of the common precursor molecule can be accounted for by three smaller peptides: a /?-lipotropin-like molecule; a corticotropin-like molecule; and a glycopeptide of unknown function (referred to as 16K fragment). The corticotropin-like segment is located in the middle of the common precursor molecule; the /3-lipotropin-like segment is located in the COOH-terminal third of the precursor (thus the /3endorphin-like segment lies at the extreme COOH-terminal end of the common precursor); 16K fragment comprises the NHS-terminal portion of the common precursor molecule. No lengthy peptide segments separate these three regions of the common precursor molecule. The ACTH biosynthetic intermediate consists of the corticotropin-like segment with 16K fragment extended from its NHz-terminal. The attachment of an oligosaccharide chain to a residue in the*COOH-terminal half of the corticotropin-like segment was previously shown to account for the structure of the smallest glycoprotein form of ACTH (Eipper, B. A., and Mains, R. E. (1977) J. BioL Chem. 252,8821-8832). Forms of the common precursor molecule and the ACTH biosynthetic intermediate that differ primarily by the addition or lack of addition of an oligosaccharide chain within their corticotropin-like segment have been identified. The structure of this common precursor molecule was determined by using a number of different techniques to examine the radiolabeled corticotropinand endorphin-containing molecules secreted by tumor cells incubated with labeled amino acids or sugars: tryptic and chymotryptic peptides of hormone fragments labeled with specific amino acids were analyzed; radioimmunoassay and immunoprecipitation techniques were used to detect ACTHand endorphin-containing fragments occurring naturally or generated by treatment with cyanogen bromide, trypsin, or chymotrypsin; gel filtration in 6 M guanidine HCl provided estimates for the molecular weights of various fragments of the common precursor.