Purification and characterization of PAM-1, an integral membrane protein involved in peptide processing.
Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the two-step alpha-amidation of peptidylglycine intermediates. PAM-1, a Type I integral membrane protein, was solubilized from the membranes of stably transfected hEK-293 cells and purified to homogeneity by antibody affinity chromatography. Purified PAM-1 exhibits an acidic pH optimum and a lower maximal velocity than soluble bifunctional PAM. Limited tryptic digestion of this integral membrane protein releases monofunctional peptidylglycine alpha-hydroxylating monooxygenase, increasing its specific activity almost fourfold and shifting its pH optimum to coincide with the pH optimum of peptidyl-alpha-hydroxyglycine alpha-amidating lyase.
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
E J
EJ
Husten
B A
BA
Eipper
D000818:Animals; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D009097:Multienzyme Complexes; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D014357:Trypsin