Differentiation of enzyme and substrate binding in the prothrombinase complex

Abstract

The Ca2+ dependence of factor Xa binding to phospholipid vesicles was measured in the presence and absence of [bovine] factor Va. The increase in polarization of a fluorescently labeled derivative of factor Xa, [5-(dimethylamino)-1-napthalenesulfonyl]glutamylglycylarginyl factor Xa (Dns-EGR-Xa), was used as a probe to measure the interaction of factor Xa with phospholipid. The Ca2+ concentration required for half-maximal binding of Dns-EGR-Xa to phospholipid vesicles was 3.5 .times. 10-4 M in the presence of factor Va and vesicles was 3.5 .times. 10-4 M in the absence of factor Va. At a Ca2+ concentration of 5 .times. 10-4 M, the binding of Dns-EGR-Xa to phospholipid-bound factor Va was near maximal, whereas there was no detectable interaction of Dns-EGR-Xa with phospholipid alone at this Ca2+ concentration as detected by fluorescence polarization. These results were qualitatively confirmed by high-performance liquid chromatography. The rate of hydrolysis of the factor Xa synthetic substrate, benzoylisoleucylglutamylglycylarginine p-nitroanilide, by factor Xa in the presence of factor Va and phospholipid decreased in a Ca2+-dependent manner. These data were analyzed as fraction of factor Xa bound to the phospholipid. A Ca2+ concentration of 2.7 .times. 10-4 M resulted in half-maximal binding by this technique. The relationship observed between rates of prothrombin activation and Ca2+ concentration could be predicted quantitatively from calculations of local enzyme and substrate concentrations.