The Activation of Prothrombin II. PARTIAL REACTIONS, PHYSICAL AND CHEMICAL CHARACTERIZATION OF THE INTERMEDIATES OF ACTIVATION

摘要: Abstract An unambiguous assignment of the product-precursor relationship is made for each step in activation prothrombin with Factor Xa. Prothrombin and its intermediates were reacted trypsin-produced Xa thrombin. [3H]Diisopropylphosphorofluoridate incorporation studies performed to identify active thrombin produced from precursors. The results obtained allow us order events process terms production previously described by this laboratory (Mann, K. G., Heldebrant, C. M., Fass, D. N. (1971) J. Biol. Chem. 246, 6106–6114). initial step, conversion Intermediate 1 3 may be catalyzed either or thrombin, however, later steps require as catalytic agent. Treatment yields two fragments which are indistinguishable those 4, a unreported fragment, concomitant 2 during cleavage 4 readily resolved DEAE-cellulose chromatography. derived citrate-Factor (Interemdiates 1, 2, 3, 4) action (Intermediates 1t 3t) have also been characterized respect their molecular weights, amino acid compositions, NH2-terminal residues. acids weights are: prothrombin-alanine, 70,000; 1-serine, 51,000; 1t-serine, 2-threonine, 41,000; 3-alanine, 23,000; 3t-alanine, 13,000. sum compositions products equivalent weight composition proposed precursor (within experimental error) every case. Intermediates 1t, 3t analysis, appear identical. Furthermore, other activators identical activation. residues permit formulation structural model indicates that proceeds sequential portions regions intermediates. These support mechanism first cleaved, yield COOH-terminal region region. then cleaved immediate precursor, contains A chain α-thrombin segment, B generate active, two-chain molecule.