The free or immobilized enzyme, in the presence of Cu ions, ascorbate, and oxygen, can be used to prepare an α-amidated protein from a polypeptide substrate possessing a carboxyl-terminal glycine residue. Glands or organs known to contain amidated peptides may contain an enzyme capable of catalyzing the amidation reaction. This may be attributed to the very low levels of enzyme present in these neuroendocrine organs. It has also been purified so as to exhibit a single, homogeneous, well-defined band following electrophoresis on sodium dodecyl sulfate/polyacrylamide gels (SDS-PAGE).
However, many mammalian proteins produced by genetic engineering technoloy require some type of post-translational processing, and this must often be accomplished by using complex, in vitro chemical or enzymatic procedures which are cost-prohibitive for large-scale production applications. 160-164 determined that rat hypothalamus also contained an α-amidating enzyme activity. The amount of the enzyme required depends on several variables well known to this art including particularly, but not limited to, the following: the specific activity of a given enzyme preparation, the amount and chemical nature of the substrate to be converted, the time within which conversion is to take place and the temperature and p H of the reaction mixture.
One type of processing activity involves the specific amidation (conversion of --COOH group to a --CONH group) of the carboxyl-terminal amino acid of a protein. 5144-5148, reported an α-amidating enzyme activity to be present in the anterior, intermediate and posterior lobes of the rat pituitary gland. Those skilled in this art will recognize other variables that may influence the precise amount of enzyme required in a given situation.
Many naturally-occurring hormones and peptides contain such a modification, which is often essential if the protein is to be biologically active. 277-281, described an α-amidating activity present in bovine pituitary neurosecretory granules. The oxygen is usually employed in stoichiometric amount but an excess of the oxygen does not affect the reaction.
An example is calcitonin, where the substitution of a non-amidated proline residue for the amidated proline of the native form results in a 3,000-fold reduction in biological activity. The presence of copper ions is also required, and can be provided by any copper salt whose anion does not adversely affect the reaction.
The agent which effects this C-terminal (alpha) amidation recognizes a glycine residue which immediately follows the amino acid to be amidated (R-X-gly, where R is the main body of the protein, X is the residue which is amidated, and "gly" is the glycine residue). 686-688 report an α-amidating enzyme activity to be present in porcine pituitary. When the enzyme has a specific enzymatic activity of about 1 m U/mg protein, maximum α-amidation occurs with a concentration of 4.7 u M cupric ions.
The glycine is cleaved and actually donates the amino moiety to the penultimate amino acid, thereby amidating it. As the purity of the enzyme is increased, the concentration requirements for the exogenous cupric ion diminishes.
Enzymatic preparations capable of amidating the carboxyl-terminus of peptides and proteins have been described from a variety of sources. The enzymatic activity can also be enhanced by the presence of ascorbate ions which can be provided by any salt, as long as the cation of the salt does not adversely effect the reaction.
Peptidyl-glycine α-amidating monooxygenase is an enzyme extractable from medullary thyroid carcinoma cell lines and tissue samples, having a molecular mass of about 60,000 to 65,000 daltons. 5144-5148; Gomez et al., FEBS Letters , Vol., 167, #1, 1984, p. Each purification step can be monitored for both protein content and the level of α-amidation activity.
It has been purified so as to exhibit a single, homogeneous, well-defined band using electrophoretic procedures... This information is used to calculate the specific activity of the enzyme which serves as an indicator of the relative purity of the enzyme.