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Stable Expression of Full-Length and Truncated Bovine Peptidylgylcine -Amidating Monooxygenase Complementary DNAs in Cultured Cells

Department of Neuroscience, Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Address requests for reprints to: Dr. Richard E. Mains, WBSB 907, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205.

Abstract

Peptidylglycine -amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the production of -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 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 -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.

FOOTNOTES

This work was supported by Grants DK-32948 and DK- 32949 from the NIH and Grants DA-00097 and DA-00098 from the NIDA. Portions of this work were previously presented as Abstract 1153 at the 70th Annual Meeting of The Endocrine Society, New Orleans, LA, 1988, and Abstract 958 at the 71 st Annual Meeting of The Endocrine Society, Seattle, WA, 1989.

Received for publication September 21, 1989. Revision received October 19, 1989. Accepted for publication October 23, 1989.

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