Identification and characterization of an amidating enzyme in ovine heart.
1. Levels of peptidylglycine alpha-amidating mono-oxygenase (PAM) activity were examined in sheep and rat heart. This enzyme is responsible for alpha-amidation of a large number of peptide hormones, a modification essential for the bioactivity of these peptides. 2. PAM activity was measured in membrane and soluble fractions of atrial and ventricular homogenates by monitoring the amidation of iodinated synthetic substrate ([125I]-Ac-Tyr-Val-Gly). 3. PAM activity in both species resided almost exclusively in the atria rather than the ventricles, and similar levels of activity were found in left and right atria. Membrane-associated activity was 50-to 100-fold greater than soluble activity in the sheep, yet was only five- to 10-fold greater in the rat, indicating a larger proportion of soluble enzyme in the rat atrium. 4. Similar apparent Km values were found for atrial membrane-associated activity in both species (15.6 and 17.4 mumol/L for rat right and left atria, 16.7 and 15.6 mumol/L for sheep right and left atria); however, the maximum velocity (Vmax) levels were higher in the rat (40.5 and 43.9 pmol/micrograms per h vs 12.8 and 15.1 pmol/micrograms per h). 5. Because expression of many peptides and processing enzymes can be regulated by steroid hormones, the possible effects of chronic glucocorticoid administration (1 mg dexamethasone i.m. twice daily for 10 days) on PAM levels were tested in four sheep, with four sheep receiving saline only as controls. There was no discernible effect of dexamethasone on either the distribution or the kinetics of PAM activity in the sheep heart.(ABSTRACT TRUNCATED AT 250 WORDS)
Lew
R A
RA
Baker Medical Research Institute, Prahran, Victoria, Australia.
Smith
A I
AI
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D019327:Copper Sulfate; D003907:Dexamethasone / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009206:Myocardium / Q000201:enzymology*; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D012756:Sheep
D009097:Multienzyme Complexes; D010455:Peptides; D003300:Copper; D003907:Dexamethasone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D019327:Copper Sulfate; D001205:Ascorbic Acid