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

Journal Article · Thu May 05 04:00:00 EDT 1988 · J. Biol. Chem.; (United States)

OSTI ID:6924966

Church, W R; Messier, T; Howard, P R; Amiral, J; Meyer, D; Mann, K G

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.

• Research Organization: Univ. of Vermont, Burlington (USA)
• OSTI ID: 6924966
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:13; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
AMINO ACID SEQUENCE
ANTIBODIES
ANTIGENS
AROMATICS
BETA DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
CALCIUM COMPOUNDS
COAGULANTS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ELEMENTS
GLOBULINS
HEMATOLOGIC AGENTS
HEPARIN ANTAGONISTS
IMMUNOASSAY
IMMUNOGLOBULINS
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAGNESIUM COMPOUNDS
MANGANESE COMPOUNDS
METALS
MOLECULAR STRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
PROTEINS
QUINONES
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RADIOISOTOPES
TRACER TECHNIQUES
TRANSITION ELEMENT COMPOUNDS
VITAMIN K
VITAMINS