Peptide C-terminal alpha-amidating enzyme purified to homogeneity from Xenopus laevis skin.

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. In this study, we have developed a simple and sensitive assay for monitoring alpha-amidating activity by using radioiodinated Ac-Tyr-Phe-Gly as a substrate. By utilizing this assay, an alpha-amidating enzyme was first purified to homogeneity from Xenopus laevis skin. The purified enzyme has a single polypeptide chain with an apparent molecular weight of 39,000 and its N-terminal sequence was determined as Ser-Leu-Ser-. The enzyme converts several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides. The enzyme activity, with an optimal pH 6-7, was dependent on the copper ion and ascorbate. In the presence of 0.25 mM ascorbate, the enzyme exhibited a Km of 0.35 microM and a Vmax of 1.9 nmol/microgram/h for Ac-Tyr-Phe-Gly.