The characterization of the ascorbic acid-mediated alpha-amidation of alpha-melanotropin in cultured intermediate pituitary lobe cells.


Previous studies have shown that cultured rat intermediate pituitary lobe cells lose the ability to form ACTH-(1-13)NH2-related molecules (alpha MSH) and instead produce ACTH-(1-14)-related peptides. In vitro studies have shown that peptidylglycine alpha-amidating monooxygenase the enzyme responsible for the conversion of ACTH-(1-14) to ACTH-(1-13)NH2, requires ascorbate, CuSO4, and molecular oxygen as cofactors. In the present study we have demonstrated that cultured intermediate pituitary lobe cells require long term supplementation of the medium with ascorbate for continued production of immunoactive alpha-amidated alpha MSH. When the relative quantities of alpha-amidated and COOH-terminally glycine-extended forms of alpha MSH were assessed in biosynthetic labeling experiments, it was shown that either L-ascorbate or an epimer of the vitamin, D-isoascorbate, was capable of supporting cellular alpha-amidation. However, the potency of isoascorbate was approximately 4- to 5-fold lower than that of ascorbate. The ascorbate-mediated reestablishment of alpha-amidation ability was shown to be dependent on the presence of sodium in the medium; at physiological levels of ascorbate (35 microM), the EC50 for sodium was about 40 mM. Time-course experiments indicated that the time of exposure of the cultured cells to ascorbate could be decreased to as little as 30 min. However, a total incubation time of 6 h was required after such an exposure to convert biosynthetically labeled ACTH-(1-14)-related peptides to labeled ACTH-(1-13)NH2-related peptides. The time course of the effects of ascorbic acid on the reestablishment of alpha-amidation, as well as the relative stereospecificity for L-ascorbic acid and the sodium requirement, are all consistent with the hypothesis that ascorbic acid must be transported into the intermediate pituitary lobe cells to participate in the peptidyl alpha-amidation reaction. Moreover, it is apparent that the length of time required to reestablish alpha-amidation ability (6 h) is a function of transport of the cofactor into the granules, the rate of the peptidylglycine alpha-amidating monooxygenase-catalyzed reaction, or both.