Functional expression and characterization of a Xenopus laevis peptidylglycine α‐amidating monooxygenase, AE‐II, in insect‐cell culture

The α‐amidating reaction of peptide hormones is a two‐step process which is catalyzed by peptidylglycine α‐hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N‐C lyase (PHL). There are three types of mRNA for these amidating enzymes in Xenopus laevis , namely AE‐I, AE‐II and AE‐III. AE‐I encodes only PHM and AE‐III encodes both PHM and PHL. AE‐II seems to encode subtypes of both PHM and PHL. While AE‐II mRNA is present in high amounts in frog skin, the actual enzymes originating from AE‐II have not been detected. When we expressed AE‐II in cultured insect‐cells using the baculovirus expression vector system, the expressed enzyme was specifically localized to the membrane fraction due to its hydrophobic transmembrane domain. Alternatively, when the transmembrane‐domain‐deleted AE‐II (Met1–Ile731) was expressed, the enzyme was secreted into the culture medium; this secreted enzyme was purified to homogeneity by a simple two‐step procedure. We have verified that the reaction product of the purified enzyme was the amidated peptide, indicating that AE‐II has the ability to catalyze the entire amidating reaction.