Peptidylglycine-alpha-amidating monooxygenase and pro-atrial natriuretic peptide constitute the major membrane-associated proteins of rat atrial secretory granules.
Peptidylglycine-alpha-amidating monooxygenase (PAM) is a bi-functional enzyme known to catalyze the post-translational bioactivation of signaling peptides. Although PAM is highly concentrated within the cardiac atrium, this tissue does not produce appreciable amounts of alpha-amidated peptides and thus, the function of PAM in atrium remains largely unknown. In this study, we demonstrate that PAM co-localizes in atrial secretory granules with the storage form of atrial natriuretic peptide (pro-ANP, amino acids 1-126), a hormone involved in the maintenance of blood pressure and fluid homeostasis. ANP is not amidated by PAM, but rather is processed to its active form (amino acids 99-126) by the proteolytic cleavage of pro-ANP. We demonstrate here by subcellular fractionation and biochemical analyses that PAM co-localizes with pro-ANP in secretory granules, where together they constitute the two most abundant membrane-associated proteins, accounting for approximately 95% of the total granular membrane protein. Respectively, light and electron microscopic immunohistochemistry show intense staining for PAM in atrial cardiomyocyctes and subcellular localization of PAM to secretory granules. Additionally, we demonstrate that while pro-ANP is readily found in the soluble contents of the granule lumen, significant amounts remain tightly associated with the membranes even after vigorous washing and estimate the molar ratio of pro-ANP to PAM to be approximately 30:1 in the membrane fraction. We postulate here that the primary function of PAM in the atrium is structural rather than enzymatic. In this regard, PAM may contribute to the packaging of pro-ANP within the secretory granule and possibly function in the presentation of pro-ANP for proteolytic processing.
O'Donnell
Patrick J
PJ
The Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA.
Driscoll
William J
WJ
Bäck
Nils
N
Muth
Elizabeth
E
Mueller
Gregory P
GP
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005260:Female; D006325:Heart Atria / Q000201:enzymology / Q000648:ultrastructure; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism* / Q000648:ultrastructure; D009097:Multienzyme Complexes / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley
D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009320:Atrial Natriuretic Factor; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase