The α-amidation of α-melanocyte stimulating hormone in intermediate pituitary requires ascorbic acid

Rat intermediate pituitary cells in primary culture display a time-dependent loss of the ability to produce COOH-terminally α-amidated αMSH. Instead of des-, mono-, and diacetyl-adrenocorticotropic hormone (ACTH)(1-13)NH₂, the cells produce des-, mono-, and diacetyl-ACTH(1-14)OH. Since the pituitary secretory granule-associated α-amidation enzyme requires copper and ascorbic acid for optimal activity, these cofactors were added to cultures of intermediate pituitary cells in an attempt to reverse the loss of peptide α-amidation ability. When the cultures were supplemented with up to 100 μM copper (II) there was very little change in the ability to α-amidate α-melanotropin. Ascorbic acid at concentrations of up to 500 μM resulted in a dramatic increase in the ability of the cells to form the α-amidated peptide. Various combinations of ascorbic acid and copper additions indicated that a relatively short exposure (hours) to ascorbic acid produced the maximal response. Ascorbic acid displayed a dose-dependent effect on the α-amidation ability with a half-optimal concentration of about 25 μM. Pulse-chase labeling experiments demonstrated the ascorbic acid-dependent conversion of labeled ACTH(1-14)OH-related peptides to ACTH(1-13)NH₂-related peptides. These results correlate with the ascorbic acid requirement of the pituitary α-amidation enzyme and demonstrate that the direct precursors to ACTH(1-13)NH₂-related peptides are ACTH(1-14)OH-related peptides. Combined with our previous data, the present studies support the notion that a wide range of neuro- and endocrine peptides become α-amidated in a similar ascorbic acid-dependent manner.