Purification and some properties of the protein component of tissue thromboplastin from human brain.
The protein component of tissue thromboplastib (Factor III) from human brain was purified by extraction of a microsomal fraction with sodium deoxycholate, gel filtration of the extract on Sephadex G-100 and preparative polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The product, apoprotein III, was homogeneous by anayltical polyacrylamide-gel electrophoresis, and it induced monospecific antibodies in rabbits and goat as shown by immunodiffusion and immunoelectrophoresis. Amino acid- and carbohydrate-analysis data for apoprotein III are presented. The carbohydrate moiety of the protein consists of fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminate, amounting to a total content of 6.3g/100g. The apoprotein alone had no procoagulant activity. When Factor III was reconstituted by combining the pure apoprotein with a purified lipid fraction from the deoxycholate extract of crude Factor III, a high and optimal procoagulant activity was obtained at a phospholipid/protein ratio of 1.1g/g. Phosphatidylethanolamine alone had a weak but significant ability to restore activity, whereas phosphatidylcholine and phosphatidylserine separately had almost none. Two-component mixtures were on average more effective, and three-component mixtures far more effective, than the single phospholipids. The inclusion of a small amount of phosphatidylserine was very important for high activity.
D000596:Amino Acids / Q000032:analysis; D001059:Apoproteins / Q000302:isolation & purification; D001923:Brain Chemistry; D002241:Carbohydrates / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010743:Phospholipids / Q000302:isolation & purification; D013925:Thromboplastin / Q000302:isolation & purification / Q000502:physiology
D000596:Amino Acids; D001059:Apoproteins; D002241:Carbohydrates; D010743:Phospholipids; D013925:Thromboplastin