Purification of -amidating enzyme and elucidation of its reaction mechanism during the maturation process of amidated peptide hormones

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A number of bioactive peptides possess a C-terminal -amide, the presence of Which in most cases is essential for their optimal bioactivities. An enzymeactivity catalyzing the -amidation reaction, peptidylglycine -amidating enzyme, was first detected in porcine pituitary in 1982 by Bradbury; now the activity is thought to be physiologically involved in the C-taminal amide formation of peptide hormones. The purposes of this study were to purify the enzyme from rat, to characterize its properties and to clarify its reaction mechanism. We preliminarily characterized the -amidating activities from rat pituitary, brain and gut, and found that these tissues, though their specific activities were different, had activities capable converting of the glycine-extended to corresponding -amidated peptides. Enzymes from these tissues had similar properties in respects of cofactor requirements and Km values fot substrates, indicating that similar enzymes are functioning in these tissues.But the crude enzymes from these tissues showed pH profile with two pH optimal peaks at neutral (6.5-7.5) and alkaline pH (8.5-9.0). Analyses by DEAE-cellulose and gel chromatographies revealed that the alkaline pH activity was due to an enzyme species of Mr of 36K (36K enzyme), on the other hand, the neutral pH activity could be elicited by combining the 36K enzyme with a protein of Mr of 41K (41K protein) which apparently showed almost no or only marginal activity at either pH 7 or 8.5. Thus, the two pH optima seen with crude enzyme were due to the presence of the two proteins at an appropriate ratio. They are found to be co-localized in the secretory vesicles wherein -amidation occurs, suggesting the combined action by these proteins being of physiological significance. The pH optimum of the -amidating enzyme has been a matter of controversy. Our finding hopefully sheds light on the problem.