PMID:8063763: Difference between revisions

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<span style = 'font-size: large'> '''Kuliopulos, A, Nelson, NP, Yamada, M, Walsh, CT, Furie, B, Furie, BC and Roth, DA'''  (1994) Localization of the affinity peptide-substrate inactivator site on recombinant vitamin K-dependent carboxylase.''J. Biol. Chem.'' '''269''':21364-70</span>
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!align=left  |Citation
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'''Kuliopulos, A, Nelson, NP, Yamada, M, Walsh, CT, Furie, B, Furie, BC and Roth, DA'''  (1994) Localization of the affinity peptide-substrate inactivator site on recombinant vitamin K-dependent carboxylase. ''J. Biol. Chem.'' '''269''':21364-70
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!align=left  |Abstract
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A recombinant His6-tagged vitamin K-dependent gamma-glutamyl carboxylase has been produced in baculovirus-infected insect cells. The His6-carboxylase shares nearly identical kinetic properties with the wild-type enzyme from bovine liver microsomes. The His6-carboxylase was irreversibly inactivated by the N-bromoacetyl-FLEEL-125I-Y peptide substrate/affinity label under pseudo-first order conditions. This inactivation could be abolished by coincubation with a high affinity peptide substrate consistent with an active site-directed inactivation. The inactivated His6-carboxylase-Ac-FLEEL-125I-Y, purified under denaturing conditions by Ni-chelation chromatography followed by preparative polyacrylamide gel electrophoresis, was subjected to proteolytic digestions with either Glu-C or Lys-C endoproteinases. The resulting polypeptide fragments were probed with three regiospecific antibodies which recognized epitopes present at the extreme N terminus (residues -23 to -13), at the hydrophobic N-terminal region (residues 86-99), and at the hydrophilic C-terminal region (residues 661-673). The site of attachment to the 125I-affinity label is located within the first 218 amino acid residues of the 758-residue carboxylase. This is the first evidence for the involvement of either the putative membrane-anchoring hydrophobic region (residues 50-314) or possibly the N-terminal hydrophilic region (residues 1-50) in gamma-carboxylation of glutamate-peptide substrates.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8063763 PubMed]


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|{{TableHeadingColor}} |'''Abstract''' || A recombinant His6-tagged vitamin K-dependent gamma-glutamyl carboxylase has been produced in baculovirus-infected insect cells. The His6-carboxylase shares nearly identical kinetic properties with the wild-type enzyme from bovine liver microsomes. The His6-carboxylase was irreversibly inactivated by the N-bromoacetyl-FLEEL-125I-Y peptide substrate/affinity label under pseudo-first order conditions. This inactivation could be abolished by coincubation with a high affinity peptide substrate consistent with an active site-directed inactivation. The inactivated His6-carboxylase-Ac-FLEEL-125I-Y, purified under denaturing conditions by Ni-chelation chromatography followed by preparative polyacrylamide gel electrophoresis, was subjected to proteolytic digestions with either Glu-C or Lys-C endoproteinases. The resulting polypeptide fragments were probed with three regiospecific antibodies which recognized epitopes present at the extreme N terminus (residues -23 to -13), at the hydrophobic N-terminal region (residues 86-99), and at the hydrophilic C-terminal region (residues 661-673). The site of attachment to the 125I-affinity label is located within the first 218 amino acid residues of the 758-residue carboxylase. This is the first evidence for the involvement of either the putative membrane-anchoring hydrophobic region (residues 50-314) or possibly the N-terminal hydrophilic region (residues 1-50) in gamma-carboxylation of glutamate-peptide substrates.
!align=left  |Keywords
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Amino Acid Sequence; Animals; Carbon-Carbon Ligases; Cattle; Cells, Cultured; Hydrolysis; Iodine Radioisotopes; Kinetics; Ligases/antagonists & inhibitors; Ligases/chemistry; Ligases/genetics; Ligases/metabolism; Molecular Sequence Data; Moths; Protein Denaturation; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Substrate Specificity
|{{TableHeadingColor}} nowrap |'''Links''' ||  [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=8063763 PubMed]
 
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Latest revision as of 04:53, 15 September 2013

Citation

Kuliopulos, A, Nelson, NP, Yamada, M, Walsh, CT, Furie, B, Furie, BC and Roth, DA (1994) Localization of the affinity peptide-substrate inactivator site on recombinant vitamin K-dependent carboxylase. J. Biol. Chem. 269:21364-70

Abstract

A recombinant His6-tagged vitamin K-dependent gamma-glutamyl carboxylase has been produced in baculovirus-infected insect cells. The His6-carboxylase shares nearly identical kinetic properties with the wild-type enzyme from bovine liver microsomes. The His6-carboxylase was irreversibly inactivated by the N-bromoacetyl-FLEEL-125I-Y peptide substrate/affinity label under pseudo-first order conditions. This inactivation could be abolished by coincubation with a high affinity peptide substrate consistent with an active site-directed inactivation. The inactivated His6-carboxylase-Ac-FLEEL-125I-Y, purified under denaturing conditions by Ni-chelation chromatography followed by preparative polyacrylamide gel electrophoresis, was subjected to proteolytic digestions with either Glu-C or Lys-C endoproteinases. The resulting polypeptide fragments were probed with three regiospecific antibodies which recognized epitopes present at the extreme N terminus (residues -23 to -13), at the hydrophobic N-terminal region (residues 86-99), and at the hydrophilic C-terminal region (residues 661-673). The site of attachment to the 125I-affinity label is located within the first 218 amino acid residues of the 758-residue carboxylase. This is the first evidence for the involvement of either the putative membrane-anchoring hydrophobic region (residues 50-314) or possibly the N-terminal hydrophilic region (residues 1-50) in gamma-carboxylation of glutamate-peptide substrates.

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Keywords

Amino Acid Sequence; Animals; Carbon-Carbon Ligases; Cattle; Cells, Cultured; Hydrolysis; Iodine Radioisotopes; Kinetics; Ligases/antagonists & inhibitors; Ligases/chemistry; Ligases/genetics; Ligases/metabolism; Molecular Sequence Data; Moths; Protein Denaturation; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Substrate Specificity

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