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J. Biol. Chem., Vol. 264, Issue 6, 3160-3168, 02, 1989

The reassociation of factor Va from its isolated subunits

S Krishnaswamy, GD Russell and KG Mann
Department of Biochemistry, University of Vermont, Burlington 05405.

Factor Va is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. The cofactor is a heterodimer composed of a light chain and a heavy chain that are associated noncovalently in the presence of divalent metal ions. The kinetics of the formation of factor Va from the isolated and separated subunits was examined by the time-dependent regain in cofactor activity using direct assays of prothrombin activation catalyzed by prothrombinase. The rate of reassociation at saturating concentrations of calcium ions was slow with a strong temperature dependence. The product of the association reaction was indistinguishable from native factor Va on the basis of activity. The second order rate constant for the process at 37 degrees C in the presence of 2 mM CaCl2 was 1.58 X 10(5) M-1.min-1. Manganese ion increased the rate of regain of activity without influencing the extent of the reaction. The previous identification of a single reactive sulfhydryl in each subunit of factor Va permitted the modification of the separated subunits with sulfhydryl-directed fluorophores. Subunit reassociation was directly measured by fluorescence energy transfer using light chain modified with 6-acryloyl- 2-dimethylaminonaphthalene (fluorescence donor) and heavy chain modified with fluorescein 5-maleimide (fluorescence acceptor). Fluorescence measurements indicate that the heavy and light chains associate tightly (Kd = 5.9 x 10(-9) M) and reversibly with a stoichiometry of 1:1. The dissociation of the subunits from the cofactor is first order with a rate constant of 1.03 X 10(-3) min-1. These interpretations were confirmed by physical measurements of subunit reassociation by sedimentation velocity studies.
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