Formation of a calcium-binding site on bovine activated factor V following recombination of the isolated subunits

Calcium binding to the activated form of blood coagulation Factor V (Factor Va) has been studied by equilibrium dialysis. A single calcium-binding site was observed with a dissociation constant of 24 +/- 4.0 microM. Unlike Factor V, Factor Va consists of two subunits. Following separation and reconstitution of the isolated subunits, a single calcium-binding site with a similar dissociation constant (24 +/- 2.0 microM) was again observed. No calcium binding was detected to either isolated subunit of Factor Va. Recombination of the Factor Va subunits used in the above equilibrium binding studies resulted in reformation of the calcium-binding site. Gel filtration experiments indicated that occupancy of this site with Ca2+ was required to maintain high affinity association between the Factor Va subunits. These data indicate that the calcium-binding site is either formed or stabilized by subunit-subunit interaction and that occupancy of this single site is sufficient to stabilize the intersubunit interactions.