Biosynthesis of amidated joining peptide from pro-adrenocorticotropin-endorphin.
Joining peptide is the major alpha-amidated product of pro-ACTH/endorphin (PAE) in AtT-20 corticotropic tumor cells. To study intracellular joining peptide synthesis, affinity purified antibodies directed against gamma-MSH, joining peptide, and ACTH were used to immunoprecipitate extracts from biosynthetically labeled AtT-20 cells. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by tryptic peptide mapping on HPLC. In steady labeling experiments, radioactivity in amidated joining peptide (JP) increased roughly linearly with time, in the manner of a final product, whereas radioactivity associated with PAE (1-94)NH2 reached a constant value after 2-4 h, indicating that PAE(1-94)NH2 is an intermediate in the biosynthesis of JP. Radioactivity appeared in ACTH(1-39) well before JP, consistent with a cleavage order in which ACTH is cleaved from PAE(1-95) before JP sequences are cleaved from PAE(1-74). This conclusion was supported by tryptic peptide analyses of immunoprecipitates, which indicated that less than 5% of JP-related material is cleaved from PAE(1-74) before being cleaved from ACTH-related sequences. After a pulse label, radioactivity in PAE(1-94)NH2 reached a peak value after 1 h of chase and declined with a half-life of less than 1 h. Amidated JP increased to a constant level after 2 h of chase. Enough radiolabeled PAE(1-94)NH2 was detected to account for about half of the radioactivity found in amidated JP, indicating that about half of JP-related material is first cleaved from PAE(1-95) before being amidated. This result was corroborated using HPLC purification to determine both amidated and glycine-extended forms of JP.
Cullen
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Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000577:Amides / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004723:Endorphins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D014316:Tritium; D014407:Tumor Cells, Cultured
D000577:Amides; D004723:Endorphins; D010446:Peptide Fragments; D011498:Protein Precursors; D014316:Tritium; D000324:Adrenocorticotropic Hormone