Gu X, Dankert J R
Department of Biology, University of Southwestern Louisiana, Lafayette 70504, USA.
J Immunol Methods. 1996 Jan 16;189(1):37-45. doi: 10.1016/0022-1759(95)00225-1.
The bactericidal activity of the C5b-9 complex of complement is dependent upon the terminal complement component C9. The precursor C5b-8 complex is not harmful to bacterial cells until C9 is added to complete the C5b-9 complex. The C9 molecule can be proteolytically cleaved by thrombin to yield an intact, nicked molecule that remains fully functional when added to either bacterial cells or erythrocytes bearing pre-formed C5b-8 complexes. In investigating the membranolytic function of C9 in the C5b-9 complex, the carboxyl-terminal portion of the nicked molecule (C9b) has been shown to be membranolytic when added to erythrocytes, liposomes, or bacterial inner membranes in the absence of any other complement components. The isolation of C9b from nicked C9 has been accomplished by preparative gel electrophoresis using detergents, however the study of the activity of C9b in membrane systems may be complicated by the possible presence of residual detergent. To address this concern, we have used 4 M urea in conjunction with hydroxyapatite chromatography and a phosphate elution procedure to separate the domains of nicked C9. The isolated C9b domain, free of detergents and in the absence of any other complement components, was found to be membranolytic. C9b isolated in this manner was capable of lysing erythrocytes and inhibiting the growth of bacterial spheroplasts.
补体C5b-9复合物的杀菌活性依赖于末端补体成分C9。在添加C9形成完整的C5b-9复合物之前，前体C5b-8复合物对细菌细胞无害。凝血酶可对C9分子进行蛋白水解切割，产生一个完整的、有切口的分子，当将其添加到带有预先形成的C5b-8复合物的细菌细胞或红细胞中时，该分子仍具有完全功能。在研究C9在C5b-9复合物中的膜溶解功能时，已表明在没有任何其他补体成分的情况下，将有切口分子的羧基末端部分（C9b）添加到红细胞、脂质体或细菌内膜时具有膜溶解作用。通过使用去污剂的制备性凝胶电泳已从有切口的C9中分离出C9b，然而，由于可能存在残留去污剂，C9b在膜系统中的活性研究可能会变得复杂。为了解决这一问题，我们使用4M尿素结合羟基磷灰石色谱法和磷酸盐洗脱程序来分离有切口C9的结构域。发现分离出的不含去污剂且没有任何其他补体成分的C9b结构域具有膜溶解作用。以这种方式分离的C9b能够裂解红细胞并抑制细菌原生质球的生长。
