Ziccardi R J
J Immunol. 1985 Apr;134(4):2559-63.
The association of native C1 with physiologically relevant proteins was studied by ultracentrifugation. 125I-C1 was centrifuged through numerous sucrose density gradients, each of which contained a different concentration of monomeric (19S) IgM throughout the gradient. The s-rate of C1 (16S) increased with increasing IgM input to a maximum of 32S. In the absence of C1q, the C1r2s2 subunit did not bind to the Ig. In gradients containing physiologic concentrations of IgM (1.3 mg/ml) at 0.14 M ionic strength, the observed s-rate of C1 was 21S. In the presence of 13 mg/ml IgG, C1 sedimented with an s-rate of 19S. Thus, under physiologic conditions, a significant fraction of native C1 is reversibly bound to monomeric Ig. SDS-PAGE analyses show that this interaction does not lead to C1 activation. The interaction of native C1 with C1 inhibitor (C1-In) was studied by ultracentrifugation at physiologic ionic strength. Purified 125I-C1-In alone sedimented with an s-rate of 4S. However in the presence of excess native C1, one-third of the C1-In co-sedimented with C1 at a 16S position. For these studies, 100 microM nitrophenylguanidinobenzoate (NPGB) was present throughout the sucrose density gradient to prevent C1 activation during centrifugation. As the concentration of NPGB was increased, the percent of 125I-C1-In at 16S decreased, indicating that C1-In was binding (reversibly) to the C1 active site region(s), which is at least partially accessible in uncleaved C1. In controls, when NPGB was omitted or activated C1 was used, the s-rate of 125I-C1-In was only 12S due to the release of C1rC1s(C1-In)2 from activated C1. Thus, under physiologic conditions native C1 is reversibly bound to C1-In.
通过超速离心研究天然C1与生理相关蛋白的关联。将125I-C1通过多个蔗糖密度梯度进行离心，每个梯度在整个梯度中含有不同浓度的单体（19S）IgM。C1（16S）的沉降速率随着IgM输入量的增加而增加，最大可达32S。在缺乏C1q的情况下，C1r2s2亚基不与Ig结合。在离子强度为0.14M、含有生理浓度IgM（1.3mg/ml）的梯度中，观察到的C1沉降速率为21S。在存在13mg/ml IgG的情况下，C1以19S的沉降速率沉降。因此，在生理条件下，相当一部分天然C1与单体Ig可逆结合。SDS-PAGE分析表明这种相互作用不会导致C1激活。在生理离子强度下通过超速离心研究天然C1与C1抑制剂（C1-In）的相互作用。纯化的125I-C1-In单独沉降时沉降速率为4S。然而，在存在过量天然C1的情况下，三分之一的C1-In在16S位置与C1共沉降。对于这些研究，在整个蔗糖密度梯度中都存在100μM硝基苯基胍苯甲酸酯（NPGB），以防止离心过程中C1激活。随着NPGB浓度的增加，16S处125I-C1-In的百分比降低，表明C1-In正在（可逆地）与C1活性位点区域结合，该区域在未裂解的C1中至少部分可及。在对照中，当省略NPGB或使用活化的C1时，由于从活化的C1中释放出C1rC1s（C1-In）2，125I-C1-In的沉降速率仅为12S。因此，在生理条件下天然C1与C1-In可逆结合。
