The membrane-bound bifunctional peptidylglycine alpha-amidating monooxygenase protein. Exploration of its domain structure through limited proteolysis.

The biosynthesis of alpha-amidated peptides from their glycine-extended precursors is catalyzed by the sequential action of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The two enzymes are part of a bifunctional, integral membrane protein precursor, peptidylglycine alpha-amidating monooxygenase (PAM). The major forms of PAM mRNA in the adult rat atrium differ by the presence or absence of optional exon A, a 315-nucleotide segment separating the PHM and PAL domains. Using antipeptide antibodies specific to the PHM, exon A, PAL, and cytoplasmic domains of rat PAM, carbonate-washed atrial membranes were found to contain proteins corresponding to rPAM-1 and rPAM-2. Digestion of atrial membranes with a variety of endoproteinases released PHM and PAL catalytic activities. Dose-response curves indicated that both catalytic activities were extremely resistant to inactivation by trypsin. Endoproteolytic digestion of atrial membranes with trypsin, chymotrypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 35-kDa PHM fragment. Digestion with trypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 42-kDa PAL fragment. In contrast to the stability exhibited by the PHM and PAL domains, the cytoplasmic domain of PAM was destroyed by most of the enzymes; only digestion with endoproteinase Lys-C generated a stable fragment. Digestion with endoproteinase Arg-C removed the carboxyl-terminal tail from PAM but failed to release the PHM or PAL domains from the membranes. The PHM fragments generated by some of the endoproteinases showed a tendency to adhere to the membranes. Thus the bifunctional PAM protein consists of independent catalytic domains separated from each other and from the putative transmembrane domain by flexible regions accessible to attack by a wide variety of endoproteinases.