Ziccardi R J
Mol Immunol. 1985 Apr;22(4):489-94. doi: 10.1016/0161-5890(85)90133-6.
The strength of interaction between the C1q and C1r2S2 subunits of C1 was studied as a function of temp. During centrifugation through sucrose density gradients at 4 degrees C, macromolecular C1 readily dissociated as it sedimented away from its free subunits. In contrast, at 20 degrees C, C1 remained associated as the 16S complex throughout centrifugation, thus indicating a stronger interaction between C1q and C1r2S2 at the higher temp. C1-inhibitor (C1-In) or nitrophenylguanidinobenzoate was present during centrifugation to prevent C1 activation. That native C1 was in fact the species being studied was confirmed by SDS-PAGE analysis. To investigate this temp dependence without using inhibitors, an alternative approach was used. Trace amounts of 125I-C1q were centrifuged through numerous sucrose density gradients, each of which contained a different concn of native C1r2S2 throughout the gradient. The s-rate of 125I-C1q increased with increasing C1r2S2 input. An association constant of 4.9 X 10(7) M-1 was calculated for this reversible interaction at 4 degrees C. However, at 20 degrees C, the data indicated a much higher affinity reaction since the addition of far less C1r2S2 was required for the s-rate of 125I-C1q to reach the 16S plateau. The presence of Cl-In did not affect these results. We have demonstrated that the association of C1q with C1r2S2 increases with increasing temp, a finding suggestive of a hydrophobic interaction. However, since we also show that C1 readily dissociates with increasing NaCl concn, the C1q-C1r2S2 interaction must, in fact, be ionic in nature. We therefore conclude that the temp dependence of the inter-subunit interaction is the result of a conformational change(s) within one of the subunits, and propose that this change may be similar to that occurring during Cl activation.
研究了补体C1的C1q与C1r2S2亚基之间相互作用的强度与温度的关系。在4℃下通过蔗糖密度梯度离心时，大分子C1在从其游离亚基沉降时很容易解离。相比之下，在20℃时，C1在整个离心过程中都保持为16S复合物的形式，这表明在较高温度下C1q与C1r2S2之间的相互作用更强。在离心过程中加入C1抑制剂（C1-In）或硝基苯基胍基苯甲酸酯以防止C1活化。SDS-PAGE分析证实所研究的确实是天然C1。为了在不使用抑制剂的情况下研究这种温度依赖性，采用了另一种方法。将微量的125I-C1q通过多个蔗糖密度梯度离心，每个梯度在整个梯度中含有不同浓度的天然C1r2S2。125I-C1q的沉降速率随着C1r2S2输入量的增加而增加。在4℃下，这种可逆相互作用的缔合常数计算为4.9×10^7 M^-1。然而，在20℃时，数据表明亲和力反应要高得多，因为要使125I-C1q的沉降速率达到16S平台所需加入的C1r2S2要少得多。C1-In的存在并不影响这些结果。我们已经证明，C1q与C1r2S2的缔合随着温度的升高而增加，这一发现提示存在疏水相互作用。然而，由于我们还表明C1随着NaCl浓度的增加而容易解离，所以实际上C1q-C1r2S2相互作用必定是离子性质的。因此我们得出结论，亚基间相互作用的温度依赖性是其中一个亚基内构象变化的结果，并提出这种变化可能类似于C1活化过程中发生的变化。
