Effect of divalent ions on the activity of cancer procoagulant

The effect of calcium (Ca2+), magnesium (Mg2+), manganese (Mn2+), iron (Fe2+), and zinc (Zn2+) on the factor X-activating activity of cancer procoagulant (CP) was studied. Activity of CP was evaluated with a three-stage chromogenic assay (liquid-phase assay, "native" CP) and with an immunocapture enzyme (ICE) assay (solid-phase assay, immobilized CP). In the liquid-phase assay, CP activity was Ca(2+)-dependent, and Mg2+ (5 mM) or Mn2+ (0.01-0.1 mM) could substitute for Ca2+. There was no additive effect of Mg2+ and Ca2+ on the activity of CP. Activity of CP in the liquid-phase assay, in the presence of 7 mM Ca2+, was enhanced by 0.1 mM Mn2+ to about 240% of the activity observed when only Ca2+ was present in the reaction. Zn2+ and Fe2+ did not activate CP in the absence of Ca2+; they inhibited CP activity in a concentration-dependent mode when administered in the presence of Ca2+. The activity of CP evaluated by the solid-phase assay (ICE assay) was neither Ca(2+)-dependent nor was it susceptible to potentiation by Mn2+ administered after CP was bound to IgM. CP exposed to 5 mM Mn2+ before binding to IgM expressed about 85% higher activity than without the presence of Mn2+. When CP was first preincubated with divalent ion and then immunocaptured, the signal generated in the enzyme-linked immunoadsorbent assay by Mn(2+)-containing CP was significantly different (30% greater) than signals generated by CP without Mn2+ or containing different ion. These data suggest that: (1) there is a significant conformational change of the CP molecule that takes place after capturing CP by the monoclonal IgM antibody on the solid surface; (2) the divalent ions are not directly involved in enzyme-substrate interactions in the CP moiety; and (3) the interaction of Mn2+ with CP seems to be different from that of the other divalent ions.