Purification and characterization of a peptidyl glycine monooxygenase from porcine pituitary.

A peptide alpha-amidating enzyme was purified to apparent homogeneity from porcine pituitary. This enzyme is a glycoprotein with a mol wt of 64,000, a metal prosthetic group, and a dependence upon ascorbate and molecular oxygen. The purified enzyme has a strong preference for peptides ending in glycine. It also catalyzes the oxidation of valylglycine bonds more rapidly than prolylglycine bonds, and demonstrates a primary isotope effect greater than 5 when the alpha-hydrogens of glycine are replaced by deuterium. Kinetic analysis is consistent with a ping-pong or double displacement catalytic mechanism in which both the peptide substrate and ascorbate are competitive inhibitors with respect to each other. With respect to its kinetic properties, catalytic mechanism, and cofactor requirements, the purified amidating enzyme is very similar to dopamine beta-hydroxylase, a finding which supports the previous suggestion that the peptide alpha-amidating enzyme be classified as a peptidyl glycine monooxygenase.

[1]  W. Busby,et al.  A rapid, sensitive assay for glycine-directed amidating enzymes , 1985, Journal of Neuroscience Methods.

[2]  W. Busby,et al.  Nonenzymatic peptide alpha-amidation. Implications for a novel enzyme mechanism. , 1985, The Journal of biological chemistry.

[3]  J. Villafranca,et al.  3-Phenylpropenes as mechanism-based inhibitors of dopamine beta-hydroxylase: evidence for a radical mechanism. , 1985, Biochemistry.

[4]  S. Miller,et al.  Secondary isotope effects and structure-reactivity correlations in the dopamine beta-monooxygenase reaction: evidence for a chemical mechanism. , 1985, Biochemistry.

[5]  W. Busby,et al.  Glycine-directed peptide amidation: presence in rat brain of two enzymes that convert p-Glu-His-Pro-Gly-OH into p-Glu-His-Pro-NH2 (thyrotropin-releasing hormone). , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[6]  W. Busby,et al.  Bovine serum albumin—GABA-His-Pro-NH2: An immunogen for production of higher affinity antisera for TRH , 1983, Journal of Neuroscience Methods.

[7]  R. Mains,et al.  Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. F. Bradbury,et al.  Substrate specificity of an amidating enzyme in porcine pituitary. , 1983, Biochemical and biophysical research communications.

[9]  S. Tate,et al.  Formation of the COOH‐terminal amide group of thyrotropin‐releasing‐factor , 1983, FEBS letters.

[10]  J. Kizer,et al.  Strategies for the preparation of haptens for conjugation and substrates for iodination for use in radioimmunoassay of small oligopeptides. , 1983, Methods in enzymology.

[11]  M. Finnie,et al.  Mechanism of C-terminal amide formation by pituitary enzymes , 1982, Nature.

[12]  J. Taylor,et al.  Purification and characterization of avian dopamine beta-hydroxylase. , 1981, Biochemistry.

[13]  S. Amara,et al.  Characterization of rat calcitonin mRNA. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Stanley N Cohen,et al.  Nucleotide sequence of cloned cDNA for bovine corticotropin-β-lipotropin precursor , 1979, Nature.

[15]  T. Spector.  Refinement of the Coomassie blue method of protein quantitation: A simple and linear spectrophotometric assay for ≤0.5 to 50 μg of protein , 1978 .

[16]  G. Kreil,et al.  Translation of melittin messenger RNA in vitro yields a product terminating with glutaminylglycine rather than with glutaminamide. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[17]  H. Morris,et al.  Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight , 1976, Nature.

[18]  J. Benjamins,et al.  Myelin synthesis during postnatal nutritional deprivation and subsequent rehabilitation , 1976, Brain Research.

[19]  B. Dobberstein,et al.  Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma , 1975, The Journal of cell biology.

[20]  A. Rich,et al.  Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue. , 1975, The Journal of clinical investigation.

[21]  S. Boguslawski,et al.  The translation of a human placental lactogen mRNA fraction in heterologous cell-free systems: the synthesis of a possible precursor. , 1975, Biochemical and biophysical research communications.

[22]  Jacob V. Maizel,et al.  Polyacrylamide Gel Electrophoresis of Viral Proteins , 1971 .

[23]  M. Goldstein,et al.  Kinetic studies of the enzymatic dopamine beta-hydroxylation reaction. , 1968, Biochemistry.

[24]  J. Whitaker.  Determination of Molecular Weights of Proteins by Gel Filtration of Sephadex. , 1963 .

[25]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.