Studies of the association of the eighth and ninth components of human complement within the membrane-bound cytolytic...

The association of the eighth (C8) and ninth (C9) components of human complement within membrane-bound C5b-9 was investigated using the photosensitive cross-linking reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Reaction of this reagent with either the purified alpha-gamma or beta subunit of C8 resulted in the introduction of 6-8 mol/mol of photosensitive 6-(4'-azido-2'-nitrophenylamino)hexanoate (ANH) as an intrinsic ligand on each protein. The resulting ANH-(alpha-gamma) or ANH-(beta) was capable of recombining with equimolar amounts of beta or alpha-gamma, respectively, to yield ANH-C8. Parallel modifications of purified C9 resulted in incorporation of 3-4 mol/mol of ANH-ligand. Both ANH-C8 and ANH-C9 retained their ability to incorporate into C5b-9. Two approaches were used to determine the proximity of C8 subunits to C9 within C5b-9. In one, the complex was assembled on erythrocytes by incubating EAC1-7 cells separately with each form of ANH-C8 and subsequently saturating with 125I-C9. After lysis, membranes were irradiated, solubilized, and analyzed by gel electrophoresis. Cross-linking was assessed by a shift in electrophoretic mobility of 125I-C9 to a higher molecular weight. Results using either form of ANH-C8 in C5b-9 showed that, although at least 30% was involved in cross-linking, none was cross-linked to C9. Similar results were obtained using a second approach in which cross-linker and radiolabel were transposed between C8 and C9. Here, EAC1-7 cells were incubated first with 125I-C8 containing either 125I-(alpha-gamma) or 125I-(beta) and subsequently with ANH-C9. Although at least 48% of ANH-C9 in C5b-9 was involved in cross-linking in these experiments, no cross-linking to either subunit of C8 was detected. These results suggest that C8 is not in close physical association with C9 within membrane-bound C5b-9.