Differential regulation of peptide alpha-amidation by dexamethasone and disulfiram.
alpha-Amidation is essential for the function of many peptides in intercellular communication. This C-terminal modification is mediated in a two-step process by the hydroxylase and lyase activities of the bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The first step, catalyzed by peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17. 3), is rate limiting in the process, and therefore subject to regulation. Dexamethasone and disulfiram (tetraethylthiuram disulfide; Antabuse) were used as in vivo treatments to study the regulation of PHM expression and activity in cardiac atrium. Our findings show that both dexamethasone and disulfiram treatment increase the activity of PHM in atrial tissue but that they do so by distinctly different mechanisms. Dexamethasone elevated tissue levels of PAM mRNA and protein concurrently, suggesting that glucocorticoids regulate PAM expression at the level of gene transcription. In contrast, disulfiram treatment, which depletes stores of alpha-amidated peptides, increased the specific activity of PHM without affecting the level of PAM expression. The catalytic efficiency of PHM was enhanced by raising the Vmax of the enzyme. Importantly, this increase in Vmax was retained through purification to homogeneity, indicating that either a covalent modification or a stable conformational change had occurred in the protein. These novel findings demonstrate that the rate-limiting enzyme in the bioactivation of peptide messengers is differentially regulated by transcriptional and post-transcriptional mechanisms in vivo. It is proposed that regulation of PHM's expression and catalytic efficiency serve as coordinated physiologic mechanisms for maintaining appropriate levels of alpha-amidating activity under changing conditions in vivo.
Driscoll
W J
WJ
Department of Physiology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
Mueller
S A
SA
Eipper
B A
BA
Mueller
G P
GP
D000818:Animals; D020134:Catalytic Domain; D003907:Dexamethasone / Q000494:pharmacology*; D004221:Disulfiram / Q000494:pharmacology*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009097:Multienzyme Complexes; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity / Q000187:drug effects
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009097:Multienzyme Complexes; D003907:Dexamethasone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D004221:Disulfiram