IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION OF THE MOLECULAR-FORMS OF APLYSIA-CALIFORNICA PEPTIDYLGLYCINE ALPHA-AMIDATING ENZYME

Peptidylglycine alpha-amidating enzyme (PAM; EC 1.14.17.3) is responsi ble for the conversion of peptides with a COOH-terminal glycine into a lpha-amidated peptides, a posttranslational modification often require d for biological activity and/or increased stability. Such an activity able to convert the model peptide D-Tyr-Val-Gly into D-Tyr-Val-amide was found to be present in the marine mollusk Aplysia californica. Exa mination of this amidating activity as well as its immunoreactivity de monstrates that (1) it can be found mainly in the atrial gland, heart, and CNS but is barely detectable in the hepatopancreas and gonads, (2 ) it requires as essential cofactors copper, molecular oxygen, and asc orbate, and (3) it exists in at least two molecular forms, a soluble a nd a membrane-bound form. Purification of this activity from the atria l gland was accomplished using Cu2+-chelating Sepharose, gel permeatio n, and hydroxyapatite chromatography, In addition, using polyclonal an tibodies raised against various parts of the rat amidating enzyme, we demonstrate that numerous immunologically recognized regions are conse rved in both the soluble and membrane-bound Aplysia californica PAM.