The alpha-amidation of alpha-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 alpha-amidated alpha MSH (Glembotski, C.C., Eipper, B.A., and Mains, R.E. (1983) J. Biol. Chem. 258, 7299-7304). 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 alpha-amidation enzyme requires copper and ascorbic acid for optimal activity (Eipper, B.A., Mains, R.E., and Glembotski, C. C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5144-5148), these cofactors were added to cultures of intermediate pituitary cells in an attempt to reverse the loss of peptide alpha-amidation ability. When the cultures were supplemented with up to 100 microM cooper (II) there was very little change in the ability to alpha-amidate alpha-melanotropin. Ascorbic acid at concentrations of up to 500 microM resulted in a dramatic increase in the ability of the cells to form the alpha-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 alpha-amidation ability with a half-optimal concentration of about 25 microM. 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 alpha-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 alpha-amidated in a similar ascorbic acid-dependent manner.