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Alpha amidating enzyme

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The remarkable similarity of the amidation reaction to that of dopamine β-monooxygenase (DBM), which converts dopamine to norepinephrine in a copper-, molecular oxygen-, and ascorbate (vitamin C)-dependent manner, suggested that ascorbate might provide the reducing equivalents essential for peptide amidation.

As predicted, ascorbate was a potent stimulator of peptide amidation catalyzed by PAM (Eipper ).

Purified enzymatic compositions are provided having alpha-amidating enzymes capable of catalyzing the conversion of a peptidyl compound having a C-terminal glycine residue to a corresponding peptidyl amide having an amino group in place of the C-terminal glycine.

Purification by size exclusion chromatography in combination with strong anion exchange chromatography results in homogeneous enzyme species which are used to prepare antibodies specific for the alpha-amidating enzyme.; A gene capable of expressing the alpha-amidating enzyme is ligated into an expression vector and transformed into a host cell capable of expressing the gene.

In order to study secretion, it was important to culture cells in a serum-free medium.

However, it soon became obvious that serum contained a factor that was essential for the conversion of α-MSH-Gly into amidated α-MSH.

The proteases that produced the longer peptides (such as pro-γ-MSH, adrenocorticotropic hormone (ACTH), joining peptide (JP), and β-lipotropin (LPH) in corticotropes) or the shorter peptides (such as α-MSH, γ-MSH, and β-endorphin in melanotropes and hypothalamic pro-opiomelanocortin (POMC) neurons) were of special interest.