Calcium-induced dissociation of human plasma factor XIII and the appearance of catalytic activity

Abstract

1. The Ca2+dependence of the activity of plasma Factor XIIIa was studied by using the continuous assay based on the incorporation of dansylcadaverine into dephosphorylated acetylated β-casein (β-substrate). The Km for Ca2+is about 0.170mm. 2. At low concentrations of Ca2+there was a lag in attaining the steady-state rate. The size of the lag was decreased and eventually abolished if the enzyme was preincubated with a high concentration of Ca2+before assay. The concentration of Ca2+required to decrease the lag phase by 50% in 10min depended on the protein concentration: at 0.87mg of protein/ml it required 17mm-Ca2+and at 0.44mg/ml it needed 10mm-Ca2+. 3. The concentrations of Ca2+required either to abolish the lag phase in the appearance of enzyme activity or to activate the essential thiol for reaction with 5,5′-dithiobis-(2-nitrobenzoate) in 10min incubation were similar at the same protein concentration. This indicated that Ca2+induces a conformation change that is responsible for both phenomena. A model is proposed that links this conformation change to the dissociation of the tetrameric enzyme. 4. This was supported by the observation that the addition of excess of b chains to the Factor XIIIa (a′2b2) increased the concentration of Ca2+required to expose the reactive thiol, and inhibited the Ca2+-dependent aggregation of a′ chains. 5. Platelet Factor XIIIa (a′2) was inhibited by 5,5′-dithiobis-(2-nitrobenzoate) in the absence of Ca2+, and no lag phases were observed in attaining the steady-state rate at low Ca2+concentrations, thus confirming the model for the activation of the plasma enzyme. 6. The Ca2+dependence of platelet Factor XIIIa indicated that Ca2+has an additional role in the enzyme mechanism of the plasma enzyme, perhaps being involved in substrate binding. 7. The dependence of the stability of plasma Factor XIIIa on Ca2+and protein concentration indicates that the decay in activity is related to the tetramer dissociation. 8. β-Substrate decreased the Ca2+concentration required for (1) abolition of the lag phase and (2) enzyme inhibition by thiol reagents. The effect on the former is greater than on the latter. 9. The role of the b chains of the plasma Factor and the evolutionary significance of the plasma and platelet Factors are considered.