Abstract
The effect of Ca2+, Mg2+, and Mn2+ on the initial rate of activation of human Factor X by the venom coagulant protein of Vipera russelli has been investigated. Neither Mg2+ nor Mn2+ alone support the reaction. Ca2+ is an essential activator and exhibits cooperative kinetics. Both Mg2+ and Mn2+ enhance the reaction cooperatively when Ca2+ is present at suboptimal concentrations. Similarly, Ca2+ quenches the intrinsic fluorescence of human Factor X in a cooperative manner. While neither Mg2+ nor Mn2+ by themselves affect the fluorescence of human Factor X, they decrease the cooperativity of the Ca2+ binding to the protein as judged by Hill plots of the Ca2+ -induced fluoresence quenching. EPR measurements indicate that there are three high affinity Mn2+ binding sites on human Factor X which can also bind Ca2+. Positive cooperativity was not observed for Mn2+ binding. These data indicate that Ca2+ can cause a conformational change of the Factor X molecule which allows the activation reaction to proceed. We propose that Mn2+ does not support the activation of human Factor X because it cannot induce a necessary conformational change in the absence of Ca2+.
Highlights
The effect of Ca’+, Mg”‘, and Mn” on the initial rate of activation of human Factor X by the venom coagulant protein of Viper russelli has been investigated
While neither M&+ nor Mn2+ by themselves affect the fluorescence of human Factor X, they decrease the cooperativity of the Ca2+ binding to the protein as judged by Hill plots of the Ca2’-induced fluorescence quenching
Since the reaction catalyzed by RVV-X does not require phospholipid, it is an ideal system to study the effect of various metal ions on Factor X activation without complications due to protein phospholipid or metal ion phospholipid interactions
Summary
From the American National Red Cross, Blood Research Laboratory, Bethesda, Maryland 20014. The effect of Ca’+, Mg”‘, and Mn” on the initial rate of activation of human Factor X by the venom coagulant protein of Viper russelli has been investigated. Extrinsic [8] blood coagulation system as well as by the coagulant protein of Vipera russelli [9] The activation of both human and bovine Factor X by these systems has an absolute requirement for Ca”+ [5, 8, 10]. Henriksen and Jackson [14] have obtained different results in that their data demonstrates that bovine Factor X binds CaZt with positive cooperativity. Since the reaction catalyzed by RVV-X does not require phospholipid, it is an ideal system to study the effect of various metal ions on Factor X activation without complications due to protein phospholipid or metal ion phospholipid interactions
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