Villiers M B, Thielens N M, Colomb M G
Biochem J. 1985 Mar 1;226(2):429-36. doi: 10.1042/bj2260429.
Soluble classical-pathway C3 convertase and proconvertase were prepared from purified C4b-C2ox complex in the presence of Ni2+; the two complexes, stable for at least 15 h at 4 degrees C, were isolated by sucrose-density-gradient ultracentrifugation. The C3 convertase alone was able to cleave C3, and its decay was accelerated in the presence of C4-binding protein. The individual roles of Ni2+ and I2 treatment of C2 in the stabilization of the complexes seemed to be different and additive. 63Ni2+ binding coupled to h.p.l.c. analysis showed that 63Ni2+ bound only to the C2ox proteolytic fragment a (1 mol/mol) with a Kd of 26 microM. Competition studies between Ni2+ and Mg2+ indicated that only half of the Ni2+ bound to the C3 convertase was removed by Mg2+, whereas, in the same conditions, Ni2+ bound to C2ox proteolytic fragment a was not displaced, suggesting the presence of two sets of sites on the convertase. EDTA prevented the formation of both C3 convertase and proconvertase; EDTA had no effect on the preformed C3 convertase, whereas it dissociated the preformed proconvertase.
在Ni2+存在的情况下，从纯化的C4b - C2ox复合物中制备可溶性经典途径C3转化酶和前转化酶；这两种复合物在4℃下至少稳定15小时，通过蔗糖密度梯度超速离心法分离。单独的C3转化酶能够裂解C3，并且在C4结合蛋白存在的情况下其衰变加速。Ni2+和I2处理C2在复合物稳定中的各自作用似乎不同且具有加和性。与高效液相色谱分析相结合的63Ni2+结合表明，63Ni2+仅以26 microM的Kd与C2ox蛋白水解片段a（1摩尔/摩尔）结合。Ni2+和Mg2+之间的竞争研究表明，只有一半与C3转化酶结合的Ni2+被Mg2+去除，而在相同条件下，与C2ox蛋白水解片段a结合 的Ni2+未被取代，这表明转化酶上存在两组位点。EDTA阻止C3转化酶和前转化酶的形成；EDTA对预先形成的C3转化酶没有影响，而它会使预先形成的前转化酶解离。
