Peptide analysis of a glycoprotein form of adrenocorticotropic hormone.

An adrenocorticotropic hormone (ACTH)-secreting mouse pituitary tumor cell line was used to investigate the structure of the smallest glycoprotein form of ACTH. AtT-20/D16v cells were incubated with a “H-amino acid or “H-sugar; “H-labeled ACTH was prepared by double-antibody immunoprecipitation of culture medium with affinity purified (~(1-24) ACTH antiserum. Immunoprecipitates were solubilized and the different forms of ACTH were separated by gel filtration. The material studied is referred to as 13K ACTH because it is precipitable with antiserum to ACTH and its apparent molecular weight on boratelacetatebuffered sodium dodecyl sulfate (SDS)-polyacrylamide gels is 13,000. Based on SDS-polyacrylamide gel electrophoresis, the preparations of 13K ACTH were over 95% pure. The apparent molecular weight of 13K ACTH in several different SDS-gel electrophoresis systems was 12,500 + 1000; molecular weight estimates were not dependent on the acrylamide content of the gels. When determined by gel filtration in 6 M guanidine HCl, the apparent molecular weight of 13K ACTH was 6700 f 600; peptide analyses indicate that the molecular weight determinations based on gel filtration in guanidine HCI are accurate. The tryptic and chymotryptic peptides of “H-labeled 13K ACTH were separated by paper electrophoresis (pH 6.35, pH 3.51, descending paper chromatography, and gel filtration in 6 M guanidine HCl. Tryptic peptides similar to each of the tryptic peptides of known (u(l-39) ACTH molecules could be identified in digests of 13K ACTH labeled with lysine, arginine, tyrosine, phenylalanine, methionine, leutine, or tryptophan; the COOH-terminal tryptic peptide of 13K ACTH is larger than the COOH-terminal tryptic peptide of known (u(l-39) ACTH molecules, but no new tryptic peptides contained in 13K ACTH, but not in (u(l-39) ACTH, were detected. Analysis of tryptic, chymotryptic, and CNBr peptides indicates that the NH,-terminal region of 13K ACTH is very similar to the NH,-terminal region of known (u(l-39) ACTH molecules. All of the carbohydrate in 13K ACTH (labeled with glucosamine, mannose, or galactose) is attached to the COOH-terminal tryptic peptide. This tryptic