Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid.
An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this alpha-amidation activity have also been found in tissue homogenates. The alpha-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; alpha-amidation activity is restored to above control levels upon addition of copper. The alpha-amidation reaction requires the presence of molecular oxygen. Of the various cofactors tested, ascorbic acid was the most potent stimulator of alpha-amidation. The alpha-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the alpha-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.
D000332:Aerobiosis; D000577:Amides / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology*; D005998:Glycine; D007700:Kinetics; D008297:Male; D010100:Oxygen; D010455:Peptides / Q000378:metabolism*; D010902:Pituitary Gland / Q000201:enzymology*; D010903:Pituitary Gland, Anterior / Q000201:enzymology; D010904:Pituitary Gland, Posterior / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D051381:Rats; D013379:Substrate Specificity; D014018:Tissue Distribution
D000577:Amides; D002614:Chelating Agents; D010455:Peptides; D003300:Copper; D001205:Ascorbic Acid; D010100:Oxygen; D005998:Glycine