title: Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation.
abstract: The COOH-terminal alpha-amidation of bioactive peptides is a 2-step process catalyzed by two separable enzymatic activities both derived from the peptidylglycine alpha-amidating monooxygenase (PAM) precursor. Two forms of PAM mRNA (rPAM-1 and -2), differing by the presence or absence of optional Exon A, were previously characterized; both encode precursors predicted to have an NH2-terminal signal sequence, an intragranular domain containing both enzymatic activities, and a single transmembrane domain followed by a short, cytoplasmic COOH-terminal domain. In this report, two novel types of PAM mRNA were identified in adult rat atrium. A cDNA of each type was sequenced, and the results indicate that rPAM-3 and -4 could be related to each other and to the previously characterized rat PAM cDNAs by alternative mRNA splicing. Deletion of a 258-nucleotide segment (optional Exon B) encoding the transmembrane domain from rPAM-3 and the presence of a novel 3'-exon in rPAM-4 mean that both rPAM-3 and -4 mRNAs encode precursor proteins that have an NH2-terminal signal peptide but lack a transmembrane domain. The rPAM-4 precursor protein lacks the region of the PAM precursor catalyzing the second step in the alpha-amidation reaction. Low levels of rPAM-3 and -4 type mRNA were detected in atrium. Utilizing the polymerase chain reaction, two major patterns of distribution of forms of PAM mRNA were found. In the heart and central nervous system, PAM mRNAs both containing and lacking optional Exon A were prevalent and almost all of the PAM mRNAs detected contained optional Exon B. In the pituitary and submaxillary glands, PAM mRNAs lacking optimal Exon A were prevalent, as were PAM mRNAs lacking all or part of optional Exon B. Since the distribution of PAM activity between soluble and membrane fractions is tissue-specific and developmentally regulated and since rPAM-4 lacks an enzymatic portion of the PAM precursor, the tissue-specific expression of these forms of rat PAM mRNA is expected to be of functional significance.
authors.affiliation: Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: D A
authors.initials: DA
authors.lastname: Stoffers
authors.forename: L
authors.initials: L
authors.lastname: Ouafik
authors.forename: B A
authors.initials: BA
authors.lastname: Eipper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016275:Atrial Function; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D015183:Restriction Mapping; D014018:Tissue Distribution
chemical_list: D009097:Multienzyme Complexes; D012333:RNA, Messenger; D004247:DNA; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase