Mielicki W P, Kozwich D L, Kramer L C, Gordon S G
University of Colorado Health Sciences Center, Denver 80262.
Arch Biochem Biophys. 1994 Oct;314(1):165-70. doi: 10.1006/abbi.1994.1425.
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.
研究了钙（Ca2+）、镁（Mg2+）、锰（Mn2+）、铁（Fe2+）和锌（Zn2+）对癌促凝素（CP）激活因子X活性的影响。采用三阶段显色测定法（液相测定法，“天然”CP）和免疫捕获酶（ICE）测定法（固相测定法，固定化CP）评估CP的活性。在液相测定中，CP活性依赖于Ca2+，Mg2+（5 mM）或Mn2+（0.01 - 0.1 mM）可替代Ca2+。Mg2+和Ca2+对CP活性无相加作用。在液相测定中，当存在7 mM Ca2+时，0.1 mM Mn2+可将CP活性提高至仅存在Ca2+时观察到的活性的约240%。在无Ca2+时，Zn2+和Fe2+不激活CP；当在Ca2+存在下给药时，它们以浓度依赖模式抑制CP活性。通过固相测定法（ICE测定法）评估的CP活性既不依赖于Ca2+，也不易被CP与IgM结合后给予的Mn2+增强。在与IgM结合之前暴露于5 mM Mn2+的CP表达的活性比不存在Mn2+时高约85%。当CP首先与二价离子预孵育然后进行免疫捕获时，含Mn2+的CP在酶联免疫吸附测定中产生的信号与不含Mn2+或含不同离子的CP产生的信号显著不同（高30%）。这些数据表明：（1）在固相表面通过单克隆IgM抗体捕获CP后，CP分子发生了显著的构象变化；（2）二价离子不直接参与CP部分中的酶 - 底物相互作用；（3）Mn2+与CP的相互作用似乎与其他二价离子不同。
