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 stab ly transfected hEK-293 cells and purified to homogeneity by antibody a ffinity chromatography. Purified PAM-1 exhibits an acidic pH optimum a nd a lower maximal velocity than soluble bifunctional PAM. Limited try ptic digestion of this integral membrane protein releases monofunction al peptidylglycine alpha-hydroxylating monooxygenase, increasing its s pecific activity almost fourfold and shifting its pH optimum to coinci de with the pH optimum of peptidyl-alpha-hydroxyglycine alpha-amidatin g lyase. (C) 1994 Academic Press, Inc.