Peptide amidation: evidence for multiple molecular forms of the amidating enzyme.

ABSTRACT

Amidating enzyme extracted from porcine pituitary was separated into glycosylated and non-glycosylated forms by fractionation on a column of Concanavalin-A Sepharose. The molecular weights of the species present were assessed by HPLC gel exclusion chromatography, which demonstrated that both the glycosylated and the non-glycosylated forms of the enzyme comprise multiple components. The apparent molecular weights of the non-glycosylated forms ranged from approximately 35 kDa to 100 kDa; the glycosylated enzyme contained species with molecular weights ranging from 65 kDa to 135 kDa. Similar proportions of glycosylated to non-glycosylated enzyme (approximately 14) were found in the anterior and posterior regions of the pituitary; higher proportions (approximately 11) were observed in the thyroid, adrenals and pancreas. The glycosylated forms of the amidating enzyme were shown to exhibit the same mandatory requirement for copper as the non-glycosylated forms, and no differences were seen in respect of their stimulation by dopamine or their pH optima. Both forms catalysed the hydroxylation of glyoxylic acid phenylhydrazone, indicating a common mechanism of action. By these criteria, glycosylation does not affect the activity of the amidating enzyme.