Kinetic analyses of peptidylglycine alpha-amidating monooxygenase from pancreatic islets.
Peptidylglycine alpha-amidating monooxygenase (PAM) plays an important role in the post-translational processing of bioactive neuropeptides by participating in C-terminal amidation. We have examined PAM activity in the pancreatic islets of the anglerfish (AF), Lophius americanus. It was previously demonstrated that the cofactor requirements and pH optimum for the fish PAM are essentially identical to PAM obtained from other tissues and species. The present study was performed to examine the enzymatic characteristics of the fish islet PAM in more detail. One of the questions addressed was the suitability of the AF islet neuropeptide Y-like peptide, aPY-Gly, as a substrate for the islet PAM. Partially purified PAM from AF islet secretory granules was incubated with [125I] aPY-Gly and the resulting products were analyzed by HPLC. The islet PAM readily mediated the formation of aPY-amide from aPY-Gly. PAM purified from bovine adrenal chromaffin granules also catalyzed the amidation of [125I] aPY-Gly. The kinetic parameters of the islet PAM were examined using trinitrophenylated-D-Tyr-Val-Gly (TNP-D-YVG) and 4-nitrohippuric acid (4-NHA). The Km of the islet PAM was 25 +/- 5 microM for TNP-D-YVG and 3.4 +/- 1 mM for 4-NHA. The competitive inhibitor of mammalian PAM activity, 4-methoxybenzoxyacetic acid, proved to be a potent inhibitor of the islet PAM as well, with an apparent KI of 0.06 mM. These results demonstrate that the AF islet PAM exhibits substrate compatibility, kinetic parameters, and inhibitor susceptibility quite similar to the characteristics of PAM from other tissues and species.
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
B D
BD
Noe
A G
AG
Katopodis
S W
SW
May
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000201:enzymology; D005399:Fishes; D007515:Islets of Langerhans / Q000201:enzymology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes
D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase