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Structure of the Precursor to an Enzyme Mediating COOH-Terminal Amidation in Peptide Biosynthesis

Department of Neuroscience, The Johns Hopkins University School of Medicine Baltimore, Maryland 21205
Endocrine Unit, Massachusetts General Hospital Boston, Massachusetts 02114
Genentech Inc. South San Francisco, California 94080

Address requests for reprints to: Dr. Betty A. Eipper, Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205.

Abstract

Many bioactive peptides terminate with an amino acid -amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine -amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary gt11 expression library. Antibodies to a β-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH₂-terminal signal sequence and short propeptide precede the NH₂ terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH₂-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.

FOOTNOTES

Supported by NIH Grants DK-32949 and DK-32948 and National Institute on Drug Abuse Grants DA-00097 and DA-00098.

Received for publication August 3, 1987. Accepted for publication September 8, 1987.

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