Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule.
mRNA was isolated from cultures of AtT-20/D-16v tumor cells and translated in a mRNA-dependent reticulocyte cell-free system. The corticotropin (ACTH) product was purified by a double-antibody immunoprecipitation procedure using antisera specific for the alpha(1-24) sequence of ACTH. The product is shown by sodium dodecyl sulfate/gel electrophoresis and gel filtration on guanidine-HCl columns to be homogeneous with an apparent molecular weight (Mr) of 28,500. A product with the same molecular weight is synthesized when membrane-bound polysomes from D-16v cells are allowed to complete their nascent chains in a reticulocyte cell-free system. Mr 31,000 ACTH isolated from tumor cells has been separated into three proteins of different apparent Mr:29,000, 32,000, and 34,000. The cell-free product contains the same lysine-, methionine-, and phenylalanine-labeled tryptic peptides as the Mr 29,000 ACTH synthesized in the tumor cells. Tryptic peptide analysis also reveals the presence of the alpha(1-39) sequence in the Mr 28,500 cell-free product and suggests that there is only one copy of this sequence in the Mr 28,500 molecule.
D000324:Adrenocorticotropic Hormone / Q000096:biosynthesis*; D000596:Amino Acids / Q000032:analysis; D002474:Cell-Free System; D055598:Chemical Phenomena; D002621:Chemistry; D066298:In Vitro Techniques; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D010446:Peptide Fragments / Q000032:analysis; D011132:Polyribosomes / Q000378:metabolism; D011233:Precipitin Tests; D012334:RNA, Neoplasm / Q000378:metabolism; D008083:beta-Lipotropin / Q000096:biosynthesis*
D000596:Amino Acids; D010446:Peptide Fragments; D012334:RNA, Neoplasm; D000324:Adrenocorticotropic Hormone; D008083:beta-Lipotropin