Hammer C H, Hänsch G, Gresham H D, Shin M L
J Immunol. 1983 Aug;131(2):892-8.
Acidification of C5 and C6 or serum to pH 6.4 at 0 degrees C, followed by neutralization, generates a factor-designated C(56)a that causes lysis of nonsensitized erythrocytes in the presence of C7, C8, and C9. C(56)a is functionally similar to alternative pathway-generated C5b,6 in respect to the formation of C5b,6,7 sites on cells, the potentiation of lytic activity by membrane-bound C3b or the membrane-active agent A2C, and the required species compatibilities between target membranes and terminal components for optimal activity. The formation of C(56)a complex from purified components C5 and C6 proceeds independently of the classical or alternative pathway C5 convertases and requires the simultaneous H+ ion treatment of the components. The generation of C(56)a from C5 and C6 and the physicochemical properties of the complex were studied in detail and compared with those of C5b,6. Acid generation of C(56)a is dose-dependent on C5 and C6 and its efficiency is similar to that of the conventional convertase in the production of lytic activity. Sucrose gradient ultracentrifugation of C(56)a containing activated 125I-C5 demonstrated a shift in sedimentation from that of native C5 to 11S, which is consistent with C5,6 complex formation. C(56)a sedimentation was identical to C5b,6, and both migrated coincident with lytic complex activity. These complexes, however, are not identical because unlike C5b,6, C(56)a is unstable at 37 degrees C, demonstrating a nonlinear decay curve. In the presence of C7, both complexes exhibit similar first order decay with a T1/2 of 3 min at 37 degrees C. SDS-PAGE autoradiographic analysis of the C5-subunit structure of 125I-C5 in C(56)a and the Zx-activated C5b,6 complex prepared from purified components showed similar alpha-chain cleavage to several fragments of 109,000, 100,000, and 58,000 daltons. Conversion to lower m.w. peptides by acid treatment was more extensive. Comparison of the 125I-C5 polypeptide chains in the membrane attack complex extracted from guinea pig erythrocyte membranes, prepared by acid activation or classical pathway lysis with whole serum, demonstrated similar C5 alpha-chain cleavage to a predominant subunit of 102,000 daltons. Acid activation also produced a 109,000 dalton C5 alpha'-fragment barely detectable with classical pathway activation. Low pH treatment of C5 alone did not inactivate C5 function, form a lytic complex on the subsequent addition of C6, or cleave the C5 alpha-chain. Thus, it is postulated that local high H+ ion concentration during simultaneous acidification of C5 and C6 allows complex formation with the concomitant C6-dependent cleavage of the C5 alpha-chain and the generation of lytic capacity.
在0℃下将C5和C6或血清酸化至pH 6.4，随后中和，会产生一种名为C(56)a的因子，该因子在C7、C8和C9存在的情况下可导致未致敏红细胞溶解。C(56)a在细胞上形成C5b,6,7位点、通过膜结合的C3b或膜活性剂A2C增强溶解活性以及靶膜与末端成分之间实现最佳活性所需的物种兼容性方面，其功能与替代途径产生的C5b,6相似。由纯化成分C5和C6形成C(56)a复合物的过程独立于经典或替代途径的C5转化酶，并且需要对这些成分同时进行H⁺离子处理。详细研究了由C5和C6生成C(56)a的过程以及该复合物的物理化学性质，并与C5b,6的性质进行了比较。C(56)a的酸生成在剂量上依赖于C5和C6，其效率与传统转化酶产生溶解活性的效率相似。对含有活化的¹²⁵I-C5的C(56)a进行蔗糖梯度超速离心，结果显示沉降从天然C5的沉降发生了变化，变为11S，这与C5,6复合物的形成一致。C(56)a的沉降与C5b,6相同，并且两者都与溶解复合物活性一起迁移。然而，这些复合物并不相同，因为与C5b,6不同，C(56)a在37℃下不稳定，呈现非线性衰减曲线。在C7存在的情况下，两种复合物在37℃下均表现出相似的一级衰减，半衰期为3分钟。对C(56)a中¹²⁵I-C5的C5亚基结构以及由纯化成分制备的Zx活化的C5b,6复合物进行SDS-PAGE放射自显影分析，结果显示α链裂解为几个分子量分别为109,000、100,000和58,000道尔顿的片段。通过酸处理转化为较低分子量的肽更为广泛。对通过酸活化或用全血清进行经典途径裂解制备的豚鼠红细胞膜中提取的膜攻击复合物中的¹²⁵I-C5多肽链进行比较，结果显示C5α链裂解为一个主要的102,000道尔顿亚基相似。酸活化还产生了一个109,000道尔顿的C5α'片段，用经典途径活化几乎检测不到。单独对C5进行低pH处理不会使C5功能失活，在随后添加C6时不会形成溶解复合物，也不会裂解C5α链。因此，据推测，在C5和C6同时酸化期间局部高H⁺离子浓度允许形成复合物，同时伴随C6依赖性的C5α链裂解并产生溶解能力。
