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Title: Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

Abstract

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of /sup 125/I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10/sup -8/ to 1 x 10/sup -6/ M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca/sup 2 +/, Mg/sup 2 +/, or Mn/sup 2 +/ partially inhibited binding of /sup 125/I-protein C to themore » antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved ..gamma..-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Vermont, Burlington (USA)
OSTI Identifier:
6924966
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.; (United States)
Additional Journal Information:
Journal Volume: 263:13
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; METALS; BIOLOGICAL EFFECTS; PROTEINS; AMINO ACID SEQUENCE; RADIOIMMUNOASSAY; ANTIBODIES; ANTIGENS; CALCIUM COMPOUNDS; ELECTROPHORESIS; IMMUNOGLOBULINS; IODINE 125; MAGNESIUM COMPOUNDS; MANGANESE COMPOUNDS; VITAMIN K; ALKALINE EARTH METAL COMPOUNDS; AROMATICS; BETA DECAY RADIOISOTOPES; COAGULANTS; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; ELEMENTS; GLOBULINS; HEMATOLOGIC AGENTS; HEPARIN ANTAGONISTS; IMMUNOASSAY; IMMUNOLOGY; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; MOLECULAR STRUCTURE; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; ORGANIC OXYGEN COMPOUNDS; QUINONES; RADIOASSAY; RADIOIMMUNOLOGY; RADIOISOTOPES; TRACER TECHNIQUES; TRANSITION ELEMENT COMPOUNDS; VITAMINS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Church, W R, Messier, T, Howard, P R, Amiral, J, Meyer, D, and Mann, K G. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding. United States: N. p., 1988. Web.
Church, W R, Messier, T, Howard, P R, Amiral, J, Meyer, D, & Mann, K G. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding. United States.
Church, W R, Messier, T, Howard, P R, Amiral, J, Meyer, D, and Mann, K G. 1988. "Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding". United States.
@article{osti_6924966,
title = {Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding},
author = {Church, W R and Messier, T and Howard, P R and Amiral, J and Meyer, D and Mann, K G},
abstractNote = {A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of /sup 125/I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10/sup -8/ to 1 x 10/sup -6/ M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca/sup 2 +/, Mg/sup 2 +/, or Mn/sup 2 +/ partially inhibited binding of /sup 125/I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved ..gamma..-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins.},
doi = {},
url = {https://www.osti.gov/biblio/6924966}, journal = {J. Biol. Chem.; (United States)},
number = ,
volume = 263:13,
place = {United States},
year = {1988},
month = {5}
}