Processing of Two Forms of the Common Precursor to α‐Melanotropin and β‐Endorphin in the Rat Pars Intermedia

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Abstract
Whole neurointermediate lobes were used to study the processing of the common precursor to β‐endorphin and α‐melanotropin (melanotropin) during pulse and pulse‐chase incubations with various radioactive amino acids. After a 30‐min pulse with radioactive amino acids, two major radioactive protein bands with apparent molecular weights of 34000 and 36000 were observed on a 10–30% acrylamide/sodium dodecylsulfate slab gel electrophoretogram. After a 2‐h chase, most of the radioactivity associated with these two protein bands had disappeared and was recovered in smaller peptides. Analysis of the tryptic fragments from the 34000‐Mr and 36000‐Mr proteins showed that both contained amino acid sequences characteristic of adrenocorticotropin (corticotropin) and β ‐lipotropin. Processing of the two precursor forms involves several proteolytic steps. The first occurring between 30 min and 1h after the start of the incubation, releases the β‐lipotropin sequence from the rest of the molecule, leaving two polypeptides with apparent molecular weights of 25000 and 27000. These two peptides are thought to have identical or closely related amino acid sequences differing only in the number of carbohydrate side chains. Analysis of the tryptic peptides of these [3H]phenylalanine‐labeled peptides showed that they contained fragments characteristic of corticotropin plus at least another fragment from the non‐corticotropin, non‐β ‐lipotropin part of the precursor molecule. Further maturation of the β‐lipotropin and the two high‐molecular‐weight forms of corticotropin occurred between 1 h and 2 h after the start of incubation. β‐Lipotropin served as a precursor for β‐endorphin while α‐melanotropin was generated by cleavage of the two high‐molecular‐weight forms of corticotropin. Peptides with apparent molecular weights of 17000 and 19000 were also recovered as major and stable end products of the maturation process. Tryptic peptide analysis showed that they were related to the portion of the precursor which does not contain either corticotropin or β‐lipotropin. When these peptides, labeled with [3H]leucine or [3H]serine, were analyzed by automated Edman degradation, it was shown that their sequence was identical in both cases to the N‐terminal sequence of the initial precursor molecule prepared during the 30‐min pulse incubation. From these data, it clearly appears that the 19000‐Mr and 17000‐Mr peptides are related to the N‐terminal fragment of the original precursor molecule.