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.