Amidating enzyme

The lower molecular mass enzyme accepts a substrate with β-alanine as the carboxyl-terminal amino acid. This invention relates to a purified α-amidating enzyme, its uses, monoclonal antibodies specific for the enzyme, and prokaryotes or other unicellular organisms or host cells isolated from multicellular organisms containing heterologous genetic material which codes for the enzyme. The human cell line HTT 54(34) has been deposited as IVI-10031. 289-293 demonstrated that D-alanine could also serve as an amino donor in the amidation reaction. The higher molecular mass species (70,000 daltons) has a specificity restricted for glycine at the carboxyl-terminus of the substrate. This and other objects of the invention will become apparent to those skilled in this art from the following detailed disclosure. Roos at the VA Medical Center in Cleveland, Ohio using human medullary thyroid carcinoma cells for the primary culture. The monoclonal antibodies allow the enzyme recovery procedures from the medullary thyroid carcinomas to be facilitated or wholly supplanted by immunoabsorption purification procedures. 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. 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. 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. This information is used to calculate the specific activity of the enzyme which serves as an indicator of the relative purity of the enzyme.

amidating enzyme-54amidating enzyme-51amidating enzyme-87amidating enzyme-6

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).

For purified enzyme having a specific enzymatic activity of approximately 50 m U/mg protein, maximal acitivity of the α-amidation occurs at about 5.5 m M ascorbate.

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.

The enzyme has been extracted from rat medullary thyroid carcinomas developed in WAG/Rij Wistar rats as described by Roos, B. A resin which may be used is the Mono Q HR5/5 strong anion exchange resin from Pharmacia Fine Chemicals and one or more passes on the column may be required for homogeneous purification of the enzyme.

It has now been discovered that homogeneously-purified α-amidating enzyme can be obtained through a multi-step procedure employing a combination of size exclusion and ion exchange chromatography from solid tumor tissue extracts, tumor cell-lines, and the tissue culture medium from such cell lines. The activity-containing eluant fraction is then subjected to ion exchange chromatography using a strong anion exchange matrix.

Leave a Reply