When guinea pig serum was treated with zymosan, hemolytic activity of C9 appeared in a macromolecular complex with a m.w. of about 1,000,000 as well as free C9. The complex was isolated by sequential gel filtrations and sucrose density gradient centrifugation. On immunodiffusion analysis the C5 and C9 incorporated into the complex showed loss of some antigenic determinants of the respective precursor proteins. Immunoelectrophoretic analysis revealed that the complex moved faster than free C9. Preliminary estimation of the subunit composition of the complex by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis showed that it contained similar proteins to those found in the human SC5b-9 complex. The complex seemed to contain one molecule each of C5b, C6, C7, C8, and protein corresponding to human S protein and three molecules of C9.

The C9 hemolytic activity of the complex was found to be very similar to that of free C9, both dose dependently and kinetically. It was neutralized with antibody to free C9 in a first order fashion, but the antibody to C5 did not neutralize the hemolytic activity in spite of its ability to precipitate the complex. The complex showed C9 hemolytic activity and also slight C7 activity. However, no other hemolytic activity was found when assayed with all the intermediate cells along the classical pathway or when assayed as any other of the components of complement. On the other hand, the human counterpart, SC5b-9, showed no C9 hemolytic activity but competitively inhibited the hemolytic activity of free C9, as reported previously by Kolb and Müller-Eberhard.

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This work was supported in part by Research Grants B-248163 and I-211306 from the Japanese Ministry of Education, Science and Culture, the Asahi Fund for Promotion of Scientific Research from the Asahi Shinbun, and a Grant for Research on Natural Science from Mitsubishi Foundation. This work was presented in part at the 52nd Annual Meeting of the Japanese Society for Bacteriology in Hiratsuka on April 5, 1979.

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