Stable expression of full-length and truncated bovine peptidylglycine alpha-amidating monooxygenase complementary DNAs in cultured cells.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the production of alpha-amidated peptides from their glycine-extended precursors, a posttranslational modification often required for full biological activity. We have previously cloned cDNAs encoding a 108-kDa bovine PAM precursor. To confirm that this cDNA encodes a functional alpha-amidating enzyme and to begin to examine the structural requirements for the biosynthesis of an active PAM enzyme, we constructed expression vectors that placed the cDNA for either the full-sized enzyme or a form truncated at the carboxyl-terminal (and thus lacking the transmembrane domain) under the control of the mouse metallothionein-1 promoter. We used the resultant plasmids to transfect AtT-20 mouse anterior pituitary corticotrope cells and selected stable lines that expressed increased levels of PAM activity. Transfected cells in which expression from the metallothionein promoter had been induced had up to 15-fold higher levels of PAM mRNA and up to 7.5-fold higher levels of PAM activity than wild-type cells. The PAM activity in the transfected cells shared many enzymatic characteristics with PAM-B, a 38-kDa soluble form of PAM purified from bovine neurointermediate pituitary. These included copper- and ascorbate-dependent activity, an alkaline pH optimum for the peptide substrate D-Tyr-Val-Gly, similar affinities for several other synthetic substrates, and comparable apparent size during gel filtration. Compared to extracts of wild-type cells, extracts from transfected cells showed increased production of five different amino acid alpha-amides. These data indicate that a single enzyme can act on a variety of peptide substrates, and that the full structure of the PAM precursor is not necessary during biosynthesis for expression of active PAM enzyme.