Peptide alpha-amidation and peptidylglycine alpha-hydroxylating monooxygenase: control by disulfiram.
The final two steps in the biosynthesis of alpha-amidated bioactive peptides are catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5). These enzymes are derived from the bifunctional precursor protein, peptidylglycine alpha-amidating monooxygenase. Because PHM is rate-limiting in peptide amidation and is copper-dependent, we examined the consequences of in vivo treatments with the copper-chelating drug disulfiram (Antabuse) on levels of alpha-amidated peptides and expression of PHM and PAL. Decreases in two amidated peptides (alpha-melanotropin and cholecystokinin) after disulfiram treatment were extremely pronounced outside the blood-brain barrier, with moderate decreases in the central nervous system. Unexpectedly, when assayed under optimal conditions in vitro, PHM activity was increased by disulfiram treatment, whereas PAL activity was unaltered. The increase in PHM activity in pituitary and atrium occurred within a few hours after the start of disulfiram treatment and was sustained up to 2 weeks after the cessation of treatment, whereas levels of alpha-amidated peptides remained low. Northern and Western blot analyses demonstrated that disulfiram had no influence on levels of peptidylglycine alpha-amidating monooxygenase mRNA or protein. Thus, inhibition of alpha-amidation by disulfiram in vivo occurs despite an increased Vmax of PHM assayed in vitro. The increase in PHM activity may result from induction of a physiologic mechanism that normally regulates this rate-limiting enzyme.
Mueller
G P
GP
Department of Physiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Husten
E J
EJ
Mains
R E
RE
Eipper
B A
BA
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism; D002766:Cholecystokinin / Q000032:analysis; D004221:Disulfiram / Q000494:pharmacology*; D004386:Duodenum / Q000378:metabolism; D006325:Heart Atria / Q000378:metabolism; D007700:Kinetics; D008297:Male; D006899:Mixed Function Oxygenases / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010455:Peptides / Q000378:metabolism*; D010902:Pituitary Gland / Q000378:metabolism; D051381:Rats; D000521:alpha-MSH / Q000032:analysis; D001615:beta-Endorphin / Q000032:analysis
D000577:Amides; D009097:Multienzyme Complexes; D010455:Peptides; D000521:alpha-MSH; D001615:beta-Endorphin; D002766:Cholecystokinin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D004221:Disulfiram