Localization of the specific binding site for magnesium(II) ions in factor IX.
We demonstrated recently that coagulation factor IX has a specific binding site(s) for Mg2+ ions, independent of the (Ca2+)-binding sites, and that binding of Mg2+ ions is very important for expression of the functional conformation of this protein. We report here the localization of this Mg2+-specific binding site. We prepared three Gla-containing fragments of bovine factor IX, namely GlaEGF(NC) (residues 1-144+286-296), GlaEGF(N) (1-83) and the Gla domain peptide (1-46). Fragments GlaEGF(NC) and GlaEGF(N) retained the ability to undergo a conformational change upon binding of Mg2+ ions in the presence of excess Ca2+ ions. This change could be detected by a conformation-specific antibody. Furthermore, the Gla domain peptide was capable of binding Mg2+ ions, as determined by the metal ion-induced quenching of the intrinsic fluorescence. It appears that the (Mg2+)-specific binding site of factor IX is located in the N-terminal Gla domain.
Sekiya
F
F
Department of Biochemistry, Meiji College of Pharmacy, Tokyo, Japan.
Yoshida
M
M
Yamashita
T
T
Morita
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002412:Cations; D002417:Cattle; D005164:Factor IX / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D002412:Cations; D010446:Peptide Fragments; D005164:Factor IX; D008274:Magnesium; D002118:Calcium