Neurosecretory Vesicles Contain Soluble and Membrane‐Associated Monofunctional and Bifunctional Peptidylglycine α‐Amidating Monooxygenase Proteins

Abstract

Peptidylglycine α-Amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of neuro-peptides in a reaction requiring the sequential action of two enzymes contained within this bifunctional protein. The CNS contains primarily transcripts encoding rPAM-1 and rPAM-2, integral membrane proteins differing by the presence or absence of a noncatalytic domain separating the two enzymes. Subcellular fractionation of adult rat hypothala-mus and hippocampus demonstrated the localization of both enzymatic activities to fractions enriched in neurose-cretory vesicles. Upon separation of the soluble contents from the membranes of neurosecretory vesicles, 30–40° of both enzymatic activities was recovered in the soluble fraction. Over 40° of both enzymatic activities remained membrane-associated following removal of peripheral membrane proteins. Antisera specific to different regions of PAM were used to identify intact rPAM-1 and rPAM-2, a monofunctional integral membrane peptidyl-α-hydroxyglycine α-Amidating lyase protein generated from rPAM-1, and a noncatalytic COOH-terminal fragment as the major PAM proteins in carbonate-washed membranes, Endopro-teolytic processing generated large amounts of soluble, monofunctional forms of both enzymes from rPAM-1 and smaller amounts of a soluble, bifunctional PAM protein from rPAM-2. A significant amount of both monofunctional enzymes lacking the transmembrane domain was tightly associated with membranes. Whereas soluble mono-and bifunctional enzymes may be released upon exocytosis of neurosecretory vesicles, membrane-associated PAM proteins may remain on the cell surface or be internalized.