The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex.
Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.
D000818:Animals; D000906:Antibodies; D002633:Chemotaxis; D002850:Chromatography, Gel; D003165:Complement System Proteins / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000276:immunology; D006461:Hemolysis; D006636:Histamine Release; D006801:Humans; D007120:Immunochemistry; D005779:Immunodiffusion; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D009504:Neutrophils; D011817:Rabbits / Q000276:immunology; D012756:Sheep / Q000276:immunology; D014461:Ultracentrifugation
D000906:Antibodies; D007457:Iodine Radioisotopes; D003165:Complement System Proteins