Functional characterization of single-chain factor X from rat liver.
14C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO4 and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. 14C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the 14C-labeled product migrated after reduction with an apparent molecular weight of 22,500 +/- 1500 (mean +/- 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin (Mr 24,500 +/- 1500; mean +/- 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight (Mr 37,000-38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the 14C. The bound 14C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a 14C-labeled polypeptide with Mr 31,000. Walker 256 tumor cells bound about 30% of the 14C. The bound material, after reduction, gave a 14C-labeled polypeptide with Mr 23,000.
D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D001780:Blood Coagulation Tests; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D007120:Immunochemistry; D008099:Liver / Q000032:analysis*; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats
D010446:Peptide Fragments; D005170:Factor X