Factor V (FV) has been isolated in homogenous form and converted from a single chain precursor protein (Mr equals 300,000) to Factor Va (FVa) (2 chains, Mr equals 115,000 and Mr equals 73,000) by limited proteolysis with thrombin. A two chain activation intermediate (FVi) (Mr equals 220,000 and 115,000) has been isolated and methods have been developed to isolate chains of both the FVi and FVa. We propose to determine the position of these chains within the FV molecule by partial amino terminal sequencing of each of the above chains. The FVa subunits have been separated by ion exchange chromatography in the presence of EDTA. Reformation of the complex with concomitant regeneration of FV activity has been obtained by incubation of the subunits in the presence of Mn2 plus or Ca2 plus. The ion binding capability of the FV, FVi, and FVa and each of the subunits will be evaluated by flow dialysis in the presence of 54Mn or 45Ca. From an evaluation of the affinity and number of ion binding sites in each of these species of FV it should be possible to gain insight into the role of these cations in maintaining the subunit-subunit interaction. Immunological properties of the subunits will be characterized in the presence and absence of Ca2 plus. Guanidino benzoate Factor Xa (GB-Xa) retains its capacity to activate prethrombin-2, but not prothrombin. GB-Xa, re-isolated from prethrombin-2 incubation mixtures, will not catalyze prothrombin activation. We propose to calculate the rate of 14CGB release from the FXa during the incubation with prethrombin-2. The kinetics of the release of the label from FXa should allow a direct demonstration of whether the deacylation rate is sufficient to allow the observed catalysis. This experiment will be repeated in the presence of FVa plus or minus fragment 2 (both of which accelerate GB-Xa activation of prethrombin-2) in order to deterimine if the deacylation rate under these conditions is adequate to account for the rate of prethrombin-2 activation observed. Finally, we will investigate the rate of 14CGB incorporation into prethrombin-2 in the presence of GB-Xa plus or minus FVa plus or minus fragment 2 to determine which, if any, of these factors facilitate formation of a catalytic site in prethrombin-2.
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