Peptidyl amidating enzyme

7-22 reported the presence of amidation signal peptides in the marine snail Apylsia. The substrate peptide or polypeptide can be purified from natural sources, synthesized from its component amino acids, or produced by recombinant DNA techniques.

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.

The chemical reaction resulting in the amidation of the carboxyl- terminus of a peptide requires a source for the amino group. DESCRIPTION OF THE INVENTION It has now been discovered that homogeneously-purified α -amidating enzyme can be obtained through a multistep 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.

5144-48, have reported that in addition to molecular oxygen, two cofactors are required for maximal enzyme activity; these are ascorbic acid and copper (II) ion. The invention further provides for the production of monoclonal antibodies to the purified enzyme and for the development of prokaryotes, other unicellular organisms or host cells isolated from multicellular organisms containing a heterologous DNA coding for peptidyl-glycine α -amidating monooxygenase. The rat cell line 77(74) was derived from rat medullary thyroid carcinoma tumors by serial passages as described by Muszynski, M. The α -amidating activity-containing product is then subjected to size exclusion chromatography on a resin of appropriate resolving capabilities, for example a Sephacryl S-200 superfine column which is available from Pharmacia Fine Chemicals.

The glycine is cleaved and actually donates the amino moiety to the penultimate amino acid, thereby amidating it. Th product, [ , was separated from the substrate by cation exchange chroma tography.

Enzymatic preparations capable of amidating the carboxyl- terminus of pep tides and proteins have been described from a. The amidating enzyme activity was also demonstrated using a synthetic substrate which mimicked the sequence of the carboxyl terminus of calcitonin, [Tyr] calcitonin (26-32).

Custom ELISA Kits, Recombinant Proteins and Antibodies can be designed, manufactured and produced according to the researcher's specifications. When the enzyme has a specific enzymatic activity of about lm U/mg protein, maximum α -amidation occurs with a concentration of 4.7 u M cupric ions. The first authors to report an approximate molecular weight for the α- amidating enzyme were Bradbury A. The presence of copper ions is also required, and can be provided by any copper salt whose anion does not adversely affect the reaction.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. The enzyme has also been sufficiently purified to permit its amino acid sequence to be determined.Many naturally-occurring hormones and peptides contain such a modification, which is often essential if the protein is to be biologically active. This information is necessary in order to permit the isolation of the genetic material coding for the enzyme and its subsequent incorporation into an appropriate unicellular organism or host cell isolated from a multicellular organism which does not contain DNA coding for the peptidylglycine α -amidating enzyme. The resulting cells containing the heterologous DNA coding for peptidyl-glycine α -amidating enzyme allows the production of sufficient quantities of the enzyme in order to perform in vitro post- translational α -amidation and theoretically permits these cells to perform this modification qf a peptide or polypeptide in vivo. The resulting enzyme is peptidyl-glycine α-amidating monooxygenase (rat source, IVI-10032; human source, IVI10033) which has a molecular mass of about 60,000 to 65,000 daltons. This information is used to calculate the specific activity of the enzyme which serves as an indicator of the relative purity of the enzyme.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. 686-688 report an α -amidating enzyme activity to be present in porcine pituitary. This is accomplished by standard recombinant DNA procedures, such as found in Maniatis, E. , et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982; or Wu, R. The α -amidation activity of the purified enzyme of this invention was demonstrated using a substrate of radioiodinated D-Tyr-Val-Gly, a peptide whose sequence mimics the carboxyl terminus of the melanocyte stimulating hormone precursor.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). Assays were performed in 100 m M TES (N-tris [hydroxymethyl] me thy 1-2-aminoe thane sulfonic acid) buffer, p H 7.0, at 37°C for three hours.It has been purified so as to exhibit a single, homogeneous, well-defined band using electrophoretic procedures performed on SDS-polyacrylamide gels, and has a specific enzymatic activity of at least 50m U per mg protein. 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 free or immobilized enzyme, in the presence of Cu 2 ions, ascorbate, and oxygen, can be used to prepare an alpha-amidated protein from a polypeptide substrate possessing a carboxyl-terminal glycine residue. For purified enzyme having a specific enzymatic activity of approximately 50 m U/mg protein, maximal activity of the α -amidation occurs at about 5.5 m M ascorbate.

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