The Experts below are selected from a list of 87 Experts worldwide ranked by ideXlab platform
D J Merkler - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine Alpha-Amidating Enzyme.
Biochemistry, 1997Co-Authors: Joseph Bell, R Kulathila, David E. Ash, Lynn M. Snyder, Ninian J. Blackburn, D J MerklerAbstract:Bifunctional peptidylglycine α-Amidating Enzyme (α-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal α-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the α-carbon of the glycyl residue, producing an α-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that α-AE requires 1.1 ± 0.2 mol of zinc/mol of Enzyme for maximal (S)-N-dansyl-Tyr-Val-α-hydroxyglycine dealkylation activity. Treatment of the Enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated Enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restora...
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Structural investigations on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine Alpha-Amidating Enzyme.
Biochemistry, 1996Co-Authors: John S. Boswell, D J Merkler, R Kulathila, Brian Reedy, Ninian J. BlackburnAbstract:The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine α-Amidating Enzyme (α-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The Enzyme contains 2 coppers per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized Enzyme indicates that 98 ± 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized Enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2−3 histidines and 1−2 O/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced Enzyme data indicates that the non-histidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands togeth...
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The irreversible inactivation of two copper-dependent monooxygenases by sulfite: peptidylglycine Alpha-Amidating Enzyme and dopamine beta-monooxygenase.
FEBS Letters, 1995Co-Authors: D J Merkler, R Kulathila, W A Francisco, D E Ash, J BellAbstract:Peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) and dopamine beta-monooxygenase (D beta M), two copper-dependent monooxygenases that have catalytic and structural similarities, are irreversibly inactivated by sodium sulfite in a time- and concentration-dependent manner. Studies with Alpha-AE show that the sulfite-mediated inactivation is dependent on the presence of redox active transition metals free in solution, with Cu(II) being the most effective in supporting the inactivation reaction. Sulfite inactivation of Alpha-AE is specific for the monooxygenase reaction of this bifunctional Enzyme and amidated peptides provide protection against the inactivation. Consequently, the sulfite-mediated inactivation of Alpha-AE and D beta M most likely results from the transition metal-catalyzed oxidation of sulfite to the sulfite radical, SO3-.
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18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine Alpha-Amidating Enzyme is a monooxygenase.
Biochemistry, 1992Co-Authors: D J Merkler, R Kulathila, Angelo P. Consalvo, Stanley D. Young, David E. AshAbstract:The biosynthesis of C-terminal Alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat Alpha-AE, we have shown that O2 is the source of the Alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[Alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [Alpha-13C]lactate generated from [Alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per Alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.
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Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglycine α-Amidating Enzyme
Archives of biochemistry and biophysics, 1992Co-Authors: D J Merkler, R Kulathila, Paul P. Tamburini, Stanley D. YoungAbstract:Conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 by recombinant type A rat 75-kDa peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) is inactivated by ascorbate, dehydroascorbate, and hydrogen peroxide in a time- and concentration-dependent manner. Both ascorbate- and dehydroascorbate-mediated inactivation are saturable with apparent kinact/Kinact values of 1.7 and 0.23 s-1 M-1, respectively. Hydrogen peroxide-mediated inactivation is not saturable with a second-order rate constant of 50 s-1 M-1. Peptidyl-Gly substrates, EDTA, and H2O2 scavengers protect against ascorbate-mediated inactivation while EDTA and semidehydroascorbate scavengers protect against dehydroascorbate-mediated inactivation. Under similar conditions, ascorbate, dehydroascorbate, and H2O2 have no effect on the Alpha-AE-catalyzed conversion of dansyl-Tyr-Val-Alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 which is consistent with the hypothesis that the 75-kDa Enzyme consists of distinct peptidyl-Gly hydroxylase and peptidyl-Alpha-hydroxyglycine lyase active sites.
Stanley D. Young - One of the best experts on this subject based on the ideXlab platform.
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Peptide substrate specificity of the α-Amidating Enzyme isolated from rat medullary thyroid CA-77 cells
International journal of peptide and protein research, 2009Co-Authors: Paul P. Tamburini, Barry N. Jones, Stanley D. Young, Randi A. Palmesino, Angelo P. ConsalvoAbstract:The kinetic parameters were obtained for enzymatic Alpha-amidation of peptides of the form N-dansyl-(Gly)4-X-Gly-OH, in which the amino acid at position X was substituted with each of the 20 natural amino acids. The Enzyme used in these studies was a highly enriched preparation of Alpha-Amidating Enzyme secreted by a clonal (CA-77) cell line which actively expresses mature Alpha-amidated peptides. A 130-fold and 11-fold variation respectively in apparent Km and Vmax values was observed. The effect of the amino acid side chain at position X in stabilization of the Enzyme-substrate complex decreased through the series X = planar aromatic or sulfur containing greater than neutral aliphatic greater than polar and basic greater than cyclic aliphatic or acidic.
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18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine Alpha-Amidating Enzyme is a monooxygenase.
Biochemistry, 1992Co-Authors: D J Merkler, R Kulathila, Angelo P. Consalvo, Stanley D. Young, David E. AshAbstract:The biosynthesis of C-terminal Alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat Alpha-AE, we have shown that O2 is the source of the Alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[Alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [Alpha-13C]lactate generated from [Alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per Alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.
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Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglycine α-Amidating Enzyme
Archives of biochemistry and biophysics, 1992Co-Authors: D J Merkler, R Kulathila, Paul P. Tamburini, Stanley D. YoungAbstract:Conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 by recombinant type A rat 75-kDa peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) is inactivated by ascorbate, dehydroascorbate, and hydrogen peroxide in a time- and concentration-dependent manner. Both ascorbate- and dehydroascorbate-mediated inactivation are saturable with apparent kinact/Kinact values of 1.7 and 0.23 s-1 M-1, respectively. Hydrogen peroxide-mediated inactivation is not saturable with a second-order rate constant of 50 s-1 M-1. Peptidyl-Gly substrates, EDTA, and H2O2 scavengers protect against ascorbate-mediated inactivation while EDTA and semidehydroascorbate scavengers protect against dehydroascorbate-mediated inactivation. Under similar conditions, ascorbate, dehydroascorbate, and H2O2 have no effect on the Alpha-AE-catalyzed conversion of dansyl-Tyr-Val-Alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 which is consistent with the hypothesis that the 75-kDa Enzyme consists of distinct peptidyl-Gly hydroxylase and peptidyl-Alpha-hydroxyglycine lyase active sites.
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Recombinant type A rat 75-kDa α-Amidating Enzyme catalyzes the conversion of glycine-extended peptides to peptide amides via an α-hydroxyglycine intermediate☆
Archives of biochemistry and biophysics, 1991Co-Authors: D J Merkler, Stanley D. YoungAbstract:The amidation of C-terminal glycine-extended peptides has been analyzed by the use of a truncated type A peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) encoded by cDNA prepared with RNA from rat medullary thyroid carcinoma (MTC) cells. Mouse C127 cells transfected with the rat MTC cDNA encoding the truncated type A Alpha-AE secrete the expected 75-kDa Enzyme into the culture medium. Medium conditioned with the transfected C127 cells converts both dansyl-Tyr-Val-Gly and dansyl-Tyr-Val-Alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 at levels which are approximately 1000 times higher than the levels found in medium conditioned with untransfected C127 cells. This result indicates that rat type A Alpha-AE alone catalyzes a two-step reaction involving an initial hydroxylation of peptidyl-Gly followed by conversion of the peptidyl-Alpha-hydroxyglycine intermediate to the amidated product. The involvement of a separate, second Enzyme to convert peptidyl-Alpha-hydroxyglycine to peptidyl-NH2 is not necessary in this system. The initial hydroxylation step is rate-determining at infinite substrate concentration and requires a reducing equivalent, molecular oxygen, and copper.
R Kulathila - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine Alpha-Amidating Enzyme.
Biochemistry, 1997Co-Authors: Joseph Bell, R Kulathila, David E. Ash, Lynn M. Snyder, Ninian J. Blackburn, D J MerklerAbstract:Bifunctional peptidylglycine α-Amidating Enzyme (α-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal α-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the α-carbon of the glycyl residue, producing an α-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that α-AE requires 1.1 ± 0.2 mol of zinc/mol of Enzyme for maximal (S)-N-dansyl-Tyr-Val-α-hydroxyglycine dealkylation activity. Treatment of the Enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated Enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restora...
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Structural investigations on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine Alpha-Amidating Enzyme.
Biochemistry, 1996Co-Authors: John S. Boswell, D J Merkler, R Kulathila, Brian Reedy, Ninian J. BlackburnAbstract:The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine α-Amidating Enzyme (α-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The Enzyme contains 2 coppers per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized Enzyme indicates that 98 ± 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized Enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2−3 histidines and 1−2 O/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced Enzyme data indicates that the non-histidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands togeth...
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The irreversible inactivation of two copper-dependent monooxygenases by sulfite: peptidylglycine Alpha-Amidating Enzyme and dopamine beta-monooxygenase.
FEBS Letters, 1995Co-Authors: D J Merkler, R Kulathila, W A Francisco, D E Ash, J BellAbstract:Peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) and dopamine beta-monooxygenase (D beta M), two copper-dependent monooxygenases that have catalytic and structural similarities, are irreversibly inactivated by sodium sulfite in a time- and concentration-dependent manner. Studies with Alpha-AE show that the sulfite-mediated inactivation is dependent on the presence of redox active transition metals free in solution, with Cu(II) being the most effective in supporting the inactivation reaction. Sulfite inactivation of Alpha-AE is specific for the monooxygenase reaction of this bifunctional Enzyme and amidated peptides provide protection against the inactivation. Consequently, the sulfite-mediated inactivation of Alpha-AE and D beta M most likely results from the transition metal-catalyzed oxidation of sulfite to the sulfite radical, SO3-.
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The Inactivation of Bifunctional Peptidylglycine α-Amidating Enzyme by Benzylhydrazine: Evidence That the Two Enzyme-Bound Copper Atoms Are Nonequivalent
Archives of biochemistry and biophysics, 1995Co-Authors: D. J. Merkler, R Kulathila, David E. AshAbstract:Peptidylglycine Alpha-Amidating Enzyme catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal Alpha-amidated peptides and glyoxylate in a reaction that requires O2, ascorbate and 2 mol of copper per mole of Enzyme [Kulathila et al. (1994) Arch. Biochem. Biophys. 311, 191-195]. Peptides with a C-terminal Alpha-hydroxyglycine residue are intermediates in the amidation reaction. Benzylhydrazine inactivates the enzymatic conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 in a time- and concentration-dependent manner. In contrast, the enzymatic conversion of dansyl-Tyr-Val-Alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 is unaffected by benzylhydrazine. The plot of 1/(inactivation rate) vs 1/[benzylhydrazine] is parabolic, indicating that the inactivation results from the interaction of 2 mol of benzylhydrazine per mole of Enzyme. EPR spectra obtained from benzylhydrazine inactivation reactions carried out in the presence of a radical trap, Alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, show the formation of a carbon-centered benzyl radical. The benzyl radical most likely results from redox chemistry between benzylhydrazine and the Enzyme-bound Cu(II) ions because EPR studies show that Enzyme-bound Cu(II) is reduced to Cu(I) in the presence of benzylhydrazine. The kinetic constants for benzylhydrazine as a reductant in the amidation reaction were determined at benzylhydrazine concentrations too low to cause significant Enzyme inactivation. Mimosine exhibits mixed inhibition vs benzylhydrazine; however, previous results have shown that benzylhydrazine is competitive vs ascorbate [Miller et al. (1992) Arch. Biochem. Biophys. 298, 380-388]. This change in kinetic mechanism coupled with the nonlinear inactivation kinetics have lead to a proposal that the two Enzyme-bound Cu(II) atoms are nonequivalent with respect to their reduction by benzylhydrazine.
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18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine Alpha-Amidating Enzyme is a monooxygenase.
Biochemistry, 1992Co-Authors: D J Merkler, R Kulathila, Angelo P. Consalvo, Stanley D. Young, David E. AshAbstract:The biosynthesis of C-terminal Alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat Alpha-AE, we have shown that O2 is the source of the Alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[Alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [Alpha-13C]lactate generated from [Alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per Alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.
David E. Ash - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine Alpha-Amidating Enzyme.
Biochemistry, 1997Co-Authors: Joseph Bell, R Kulathila, David E. Ash, Lynn M. Snyder, Ninian J. Blackburn, D J MerklerAbstract:Bifunctional peptidylglycine α-Amidating Enzyme (α-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal α-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the α-carbon of the glycyl residue, producing an α-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that α-AE requires 1.1 ± 0.2 mol of zinc/mol of Enzyme for maximal (S)-N-dansyl-Tyr-Val-α-hydroxyglycine dealkylation activity. Treatment of the Enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated Enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restora...
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The Inactivation of Bifunctional Peptidylglycine α-Amidating Enzyme by Benzylhydrazine: Evidence That the Two Enzyme-Bound Copper Atoms Are Nonequivalent
Archives of biochemistry and biophysics, 1995Co-Authors: D. J. Merkler, R Kulathila, David E. AshAbstract:Peptidylglycine Alpha-Amidating Enzyme catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal Alpha-amidated peptides and glyoxylate in a reaction that requires O2, ascorbate and 2 mol of copper per mole of Enzyme [Kulathila et al. (1994) Arch. Biochem. Biophys. 311, 191-195]. Peptides with a C-terminal Alpha-hydroxyglycine residue are intermediates in the amidation reaction. Benzylhydrazine inactivates the enzymatic conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 in a time- and concentration-dependent manner. In contrast, the enzymatic conversion of dansyl-Tyr-Val-Alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 is unaffected by benzylhydrazine. The plot of 1/(inactivation rate) vs 1/[benzylhydrazine] is parabolic, indicating that the inactivation results from the interaction of 2 mol of benzylhydrazine per mole of Enzyme. EPR spectra obtained from benzylhydrazine inactivation reactions carried out in the presence of a radical trap, Alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, show the formation of a carbon-centered benzyl radical. The benzyl radical most likely results from redox chemistry between benzylhydrazine and the Enzyme-bound Cu(II) ions because EPR studies show that Enzyme-bound Cu(II) is reduced to Cu(I) in the presence of benzylhydrazine. The kinetic constants for benzylhydrazine as a reductant in the amidation reaction were determined at benzylhydrazine concentrations too low to cause significant Enzyme inactivation. Mimosine exhibits mixed inhibition vs benzylhydrazine; however, previous results have shown that benzylhydrazine is competitive vs ascorbate [Miller et al. (1992) Arch. Biochem. Biophys. 298, 380-388]. This change in kinetic mechanism coupled with the nonlinear inactivation kinetics have lead to a proposal that the two Enzyme-bound Cu(II) atoms are nonequivalent with respect to their reduction by benzylhydrazine.
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18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine Alpha-Amidating Enzyme is a monooxygenase.
Biochemistry, 1992Co-Authors: D J Merkler, R Kulathila, Angelo P. Consalvo, Stanley D. Young, David E. AshAbstract:The biosynthesis of C-terminal Alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine Alpha-Amidating Enzyme (Alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat Alpha-AE, we have shown that O2 is the source of the Alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[Alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [Alpha-13C]lactate generated from [Alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per Alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.
Roger Acher - One of the best experts on this subject based on the ideXlab platform.
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Evidence for distinct dibasic processing endopeptidases with Lys-Arg and Arg-Arg specificities in neurohypophysial secretory granules
Biochemical and Biophysical Research Communications, 1992Co-Authors: Y. Rouillé, Anne Spang, Jacqueline Chauvet, Roger AcherAbstract:Two Ca(2+)-dependent endopeptidases endowed with specificities for paired basic residues have been disclosed in rat and ox neurohypophysial secretory granules. Specificities investigated by using synthetic fluorogenic substrates showed the presence of a Lys-Arg endopeptidase with optimum pH close to the granule pH (5.5) and of an Arg-Arg endopeptidase more active at pH 7.0. Granule extracts have virtually no activity towards Lys-Lys-containing substrate or monobasic substrates. Pro-Gly-Lys-Arg-chloromethylketone appears a very efficient inhibitor for the Lys-Arg Enzyme. Soluble and membrane-bound forms of both endopeptidases have been detected. pH-dependence of membrane binding and partitioning into Triton X-114 suggest that the membrane-bound form of Lys-Arg endopeptidase is associated through an amphiphilic Alpha-helix. It is proposed that the Enzyme Lys-Arg cleaves prooxytocin and provasopressin at their signal sequence Gly-Lys-Arg when these precursors arrive in the neurosecretory granules. The processing proceeds in the granules through carboxypeptidase E and Alpha-Amidating Enzyme complex for giving mature pharmacologically active nonapeptide hormones.
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Heterologue Conversion of Amphibian Hydrin 2 into Vasotocin Through Bovine Granule Alpha-Amidating Enzyme
Journal of neuroendocrinology, 1991Co-Authors: Y. Rouillé, Jacqueline Chauvet, Roger AcherAbstract:Hydrin 2 (vasotocinyl-Gly), found along with vasotocin in the neurohypophysis of frogs and toads but not of other vasotocin-bearers such as birds and reptiles, is believed to act on water permeability of frog skin, whereas vasotocin mainly fulfils the antidiuretic function on the kidney. In order to understand the peculiar regulation of provasotocin differential processing in amphibians, conversion of hydrin 2 into vasotocin has been attempted using bovine pituitary granule α-Amidating Enzyme. Generated vasotocin has pharmacological properties and Chromatographic behaviour in high-performance liquid chromatography identical to those of synthetic vasotocin. However, the low yield of conversion (5% to 10%) suggests that additional factor(s) might be involved in the physiological processing.