Structural transitions in bovine factor X associated with metal binding and zymogen activation. Studies using conformation-specific antibodies.
Conformation-specific antibodies against distinct regions of Factor X were employed to locate antigenic determinants which are altered during zymogen activation or by metal binding. Anti-Factor X antibodies, raised in rabbits against Factor X, were purified by affinity chromatography using Factor X covalently bound to Sepharose. Quantitative equilibrium and kinetic measurements of precipitation of Factor X and Factor Xa by antibodies indicated differences in the antigenic structure of the zymogen and the enzyme form of factor X. The factor X antibodies were further fractionated by sequential immunoabsorption using fragments of Factor X and Factor Xa. With conformation-specific antibodies directed against the heavy chain and the light chain of Factor X, zymogen activation was shown to involve a structural transition in the heavy chain but not the light chain. Antibodies directed against the activation peptide domain 1-51 of the heavy chain, the trypsin-like region of the heavy chain 52-290, and the substrate-binding site suggest a generalized conformational transition in the heavy chain. Antibodies were isolated which are specific for the Factor X:Ca(II) complex and bind to Factor X only in the presence of metal ions. Subfractions were directed against either the heavy chain or the light chain, indicating that both the heavy chain and the light chain of Factor X undergo a metal-induced conformational transition. Half-maximal antibody-factor X interaction was observed at 0.13 mM CaCl2 for the light chain and 0.7 mM CaCl2 for the heavy chain. These results indicate that zymogen activation is limited to structural changes in the heavy chain, but metal binding is associated with changes in the structure of both the heavy and light chains. Metal-dependent binding of Factor X to the platelet Factor Xa receptor after activation may involve surfaces of the heavy as well as the light chains.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride; D002417:Cattle; D002846:Chromatography, Affinity; D000939:Epitopes / Q000276:immunology*; D005170:Factor X / Q000276:immunology / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000939:Epitopes; D008670:Metals; D010446:Peptide Fragments; D005170:Factor X; D002122:Calcium Chloride; D002118:Calcium