Immunochemical analyses of membrane-bound complement. Detection of the terminal complement complex and its similarity...

(1) Membranes of sheep erythrocytes lysed with antibody and human or rabbit complement were solubilized in non-ionic detergents (Triton X-100 or Berol EMU-043) and analysed immunochemically using antisera directed against individual complement components. The precipitation behaviour of membrane-bound C3, C5, C6 and C9 components of complement was examined by immuno-double diffusion, rocket- and crossed immunoelectrophoresis performed in agarose gels containing 1% non-ionic detergent. (2) Membrane-bound C5, C6 and C9 are antigenically altered compared with the native (serum) components. (3) Immuno-double diffusion in the presence of non-ionic detergents reveals formation of C5-C6-C9 complexes on the membranes; these complexes are stable in non-ionic detergent. No complex formation was detected in serum between native C5, C6 and C9 components. There was also no evidence for complexing between membrane-bound C3, C4 or membrane proteins and the "late-reacting" complement components. (4) The extractability of complement components by various manipulations has been studied by use of quantitative rocket immunoelectrophoresis. Up to 65% of membrane-bound C3 is readily extracted by dialysis of membranes against 1mM EDTA, pH 8.0, 100 mM EDTA, pH 8.0, 1.2 NaCl plus or minus EDTA, by extraction in isotonic buffers at 37 degrees C, by heating at 45 degrees C over several hours, or by treating membranes with 1 mM p-chloromercuribenzoate sulfonate. In contrast, less than 6% of the terminal complement complex can be eluted by any of the described methods or combination of methods. (5) Our data suggest that the terminal complement complex associates with membrane "core" components through apolar interactions.