Peptidyl amidating enzyme updating windows key
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.
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 peptidyl-glycine α-amidating enzyme.
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.
This information is used to calculate the specific activity of the enzyme which serves as an indicator of the relative purity of the enzyme.
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.
3228-3232, showed two distinct enzyme activities in rat brain which were capable of catalyzing the α-amidating reaction. It is therefore the object of the invention to provide a purified α-amidating enzyme which can efficiently be used to produce α-amidated peptides from peptide or polypeptide substrates, to prepare monoclonal antibodies specific for the enzyme, and to construct prokaryotes or other appropriate unicellular organisms or host cells isolated from multicellular organisms containing heterologous DNA coding for the enzyme.
The chemical reaction resulting in the amidation of the carboxyl-terminus of a peptide requires a source for the amino group. and p H 7.0.] The invention also provides a method of preparing an α-amidating peptide from peptide or polypeptide substrates containing a terminal glycine residue by reacting the substrate with oxygen in the presence of the free or immobilized purified enzyme, ascorbate and copper. The enzyme has also been extracted from other sources, notably human and rat medullary thyroid carcinoma cell lines. The sample, for example, can be bulk-loaded on a preparative scale anion exchange column such as a DE-52 resin from Whatman, Limited.