Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid.

Abstract

An enzymatic activity capable of producing an .alpha.-amidated peptide product from its glycine-extended precursor was 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 Cu. The .alpha.-amidation reaction requires the presence of molecular O2. 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.