Evidence of direct insertion of terminal complement proteins into cell membrane bilayers during cytolysis. Labeling by a photosensitive membrane probe reveals a major role for the eighth and ninth components

Radioiodinated hexanoyldiiodo-N-(4-azido-2-nitrophenyl)tyramine (HNT) was employed as a photosensitive membrane-restricted probe to establish whether terminal complement proteins insert into membrane bilayers during cytolysis. The system studied consisted of natural membranes carrying intermediate (C5b-8) or fully assembled (C5b-9) cytolytic complexes of human complement prepared by two different methods. In one method, C5b-8 and C5b-9 were assembled on membranes de novo by incubating rabbit erythrocytes with C9-depleted or whole human serum, respectively. The probe was partitioned into lipid bilayers of the resulting MC5b-8 and MC5b-9 membranes by post-addition of HNT. Membranes were irradiated and photolabeled C5b-8 and C5b-9 were extracted, purified, and analyzed on polyacrylamide gels. Those constituents labeled by HNT within each complex were identified by autoradiography. The second method involved pre-addition of HNT to membranes carrying the precursive C5b-7 complex and subsequent conversion to MC5b-8 and MC5b-9 by addition of exogenous C8 and C9. After irradiation, C5b-8 and C5b-9 were again purified and analyzed for the presence of photolabel. Results from both methods were similar and indicated all constituents of each complex are labeled to a measurable extent. However, the C8 alpha subunit was predominantly labeled in C5b-8 and both C8 alpha and C9 were predominantly labeled in C5b-9. Because labeling by HNT is specific for intramembrane structural domains of proteins, these results provide direct evidence that constituents of terminal complement complexes insert into the lipid bilayer of cell membranes during lysis. Further, we conclude that in these complexes, C8 alpha and C9 are the primary contributors of inserted peptide domains.