Abstract
The binding isotherms of Ca2+ and Mn2+ to bovine factor IX have been determined at pH 6.5 and pH 7.4, at 25 degrees C. At pH 7.4, at least 2 strong Ca2+ sites, with an average KDISS of 0.1 +/- 0.02 mM, are found. An additional 10 to 11 weaker Ca2+ binding sites, with an average KDISS of 1.3 +/- 0.2 mM are noted, at high levels of Ca2+. At pH 6.5, again at least 2 strong Ca2+ sites on factor IX are evident, with an average KDISS of 0.11 +/- 0.02 mM; but slightly fewer (7 to 8) weaker sites, with an average KDISS of 1.9 +/- 0.3 mM, are obtained. Qualitatively, the binding of Mn2+ to bovine factor IX appears similar to that of Ca2+. At pH 6.5, approximately 2 strong Mn2+ binding sites, with an average KDISS of 13 +/- 1.5 micrometer, and an additional 5 to 6 weak sites, with an average KDISS of 160 +/- 15 micrometer, are present. Manganese does not completely displace Ca2+; and Ca2+ does not completely displace Mn2+ from their respective binding sites. On the other hand, Tb3+ and Sm3+ readily displace Ca2+, at pH 6.5, from its sites on factor IX. The rate and extent of activation of bovine factor IX, by bovine factor XIa, is dependent on the Ca2+ concentration, up to concentrations of Ca2+ which saturate its effect on the system. Substitution of Sr2+ for Ca2+ leads to approximately 50% of the maximum rate of factor IXa formation, and final yield of factor IXa, in this activation system. Manganese does not substitute for Ca2+ in this activation, but does inhibit the stimulatory effect of Ca2+. Both Tb3+ and Sm3+ are effective inhibitors of Ca2+ in factor IX activation by factor XIa.
Highlights
The binding isotherms of Ca2+ and Mn” to bovine factor IX have been determined at pH 6.5 and pH 7.4, at 25°C
The rate and extent of activation of bovine factor IX, by bovine factor XIa, is dependent on the Ca2+ concentration, up to concentrations of Ca2+ which saturate its effect on the system
At pH 6.5, again, at least 2 strong Ca2+ binding sites are noted, with an average KDM of 0.11 f 0.02 mM, and an additional 7 to 8 weak sites, with an average KDISS of 1.9 f 0.3 IIIM are calculated by experimental curve fitting of the data of Fig. 1B
Summary
(Received for publication, December 30, 1977, and in revised form, March 9, 1978). From the Department of Chemistry, The University of Notre Dame, Notre Dame, Indiana 46556. Since the vitamin Kdependent portion of factor IX, present on its NHz-terminal region [12, 13], is conserved after activation to factor IXa [2], it can be logically assumed that factor IXa possesses important Ca2+ binding properties. This is in contrast to prothrombin, which loses its vitamin K-dependent portion as a consequence of its activation to thrombin [14, 15]. We report results which we obtained in a study of the metal binding properties of this important zymogen
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