Peptidyl amidating enzyme
and p H 7) and being substantially free of other proteolytic enzymes which degrade the peptide, the polypeptide, the amidated peptide or the amidated polypeptide, or PAM, wherein the purified enzyme preparation is prepared by a process comprising: (a) subjecting a PAM containing composition obtained from a medullary thyroid carcinoma tissues, cell lines thereof, or tissue culture media from such cell lines to anion exchange chromatography and then size exclusion chromatography to produce a PAM containing eluant fraction; and (b) subjecting the eluant fraction to strong anion exchange chromatography to produce the purified enzyme preparation. The method of claim 1, wherein the enzymatic activity is at least 50/m U/mg protein. The method of claim 2, wherein the purified enzyme preparation is homogeneous. The method of claim 1, 2, or 3, wherein the medullary thyroid carcinoma is derived from a rat. The method of claim 4, wherein the rat medullary thyroid carcinoma is deposit IVI-10028, and the cell line is deposit IVI-10029. The method of claim 1, 2, or 3, wherein the PAM has a molecular mass of 60,000 to 65,000 daltons. The method of claim 2 or 3, wherein the purified preparation exhibits a single, homogeneous, well defined band following electrophoresis on SDS/polyacrylamide gel. For example, lower life forms such as the dog fish (Squalus acanthias) have been reported by O'Donohue T. 353-395, to contain amidated peptides in pituitary extracts. Heretofore, the purification and characterization of the α-amidating enzyme have not been published. Using Sephadex G-100 they suggested a minimum apparent molecular mass of approximately 60,000 daltons. The purified peptidyl-glycine α-amidating monooxygenase is used to amidate the alpha-carboxyl group of a polypeptide having a terminal glycine residue, where the glycine functions as an amino group donator.
A method of preparing an alpha-amidated peptide or polypeptide from a peptide or polypeptide substrate having a terminal glycine residue having an alpha-carboxyl group comprising reacting said substrate in the presence of an enzymatically effective amount of a purified enzyme preparation containing peptidyl-glycine alpha-amidating monooxygenase, PAM, said substrate purified from natural sources or produced by recombinant DNA techniques and said enzyme preparation capable of amidating the alpha-carboxyl group of the substrate, said preparation having a specific enzymatic activity of at least 25 m U/mg protein (as measured by the conversion of Dansyl-D-Tyr-Val-Gly-COOH to Dansyl-D-Tyr-Val-CONH at 37° C. 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).
Since the peptidyl-glycine α-amidating monooxygenase has been sufficiently purifed, it is now possible to obtain monoclonal antibodies directed against the enzyme by standard procedures. 1522-1530, reported that a murine cell line derived from the anterior pituitary lobe (ATT-20) contained an α-amidating enzyme activity that apparently decreased with time in culture. Despite the apparent ubiquitous distribution of this activity in nature, little information has been published on its physicochemical characteristics. It has been purified such that it exhibits a specific enzymatic activity (number of units of α-amidation activity per milligram of protein) of at least approximately 25 m U and preferably at least approximately 50 m U/mg protein. 7-22 reported the presence of amidation signal peptides in the marine snail Apylsia. The resulting enzyme is peptidyl-glycine α-amidating monooxygenase (deposited rat derived enzyme, IVI-10032; deposited human derived enzyme, IVI-10033) which has a molecular mass of about 60,000 to 65,000 daltons. 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. 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.