Regulation of peptide amidation in cultured pituitary cells.
The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.
D000818:Animals; D001205:Ascorbic Acid / Q000032:analysis; D001971:Bromocriptine / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D003300:Copper / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D009074:Melanocyte-Stimulating Hormones / Q000096:biosynthesis; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010902:Pituitary Gland / Q000166:cytology / Q000201:enzymology*; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D051381:Rats; D012701:Serotonin / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D009097:Multienzyme Complexes; D012701:Serotonin; D001971:Bromocriptine; D005680:gamma-Aminobutyric Acid; D003994:Bucladesine; D003300:Copper; D009074:Melanocyte-Stimulating Hormones; D003346:Corticotropin-Releasing Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D007545:Isoproterenol; D001205:Ascorbic Acid