Measurement of the ratio of the eighth and ninth components of human complement on complement-lysed membranes.
The mole ratio of the eighth (C8) and ninth (C9) components of human complement on membranes carrying the cytolytic C5b-9 complex was measured by direct binding assays. Erythrocytes from two different species were used as the membrane system. Antibody-treated sheep erythrocytes carrying a relatively small number of precursive membrane-bound C5b-7 complexes were prepared by exposure to human C8-depleted serum. These complexes were subsequently converted to C5b-8 by addition of saturating amounts of C8. Parallel binding assays using 125I-C8 were used to determine the exact amount bound and thus the number of C5b-8 complexes per cell. These cells were subsequently incubated with excess 125I-C9 and the amount bound relative to C8 on the membrane was measured. Results indicated the C8:C9 ratio remained constant at approximately 1:4 as the number of complexes varied from 40 to 310 per cell. Similar results were obtained regardless of whether C8 and C9 were added sequentially or simultaneously to cells bearing C5b-7. For comparison, experiments were also performed using membranes that contained a high number of complexes. Here, rabbit erythrocytes which carried approximately 25 000 C5b-7 per cell were incubated with limited amounts of C8 to form C5b-8 complexes on the membrane surface, the exact number of which was measured by 125I-C8 binding assays. When erythrocytes prepared in this manner were incubated with excess 125I-C9, the ratio of C8:C9 on the membrane was found to be essentially constant at approximately 1:3 as the number of these complexes varied from 50 to 4000 per cell.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001667:Binding, Competitive; D003185:Complement C8 / Q000378:metabolism*; D003186:Complement C9 / Q000378:metabolism*; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000032:analysis*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D011817:Rabbits; D011951:Receptors, Complement / Q000378:metabolism*; D012756:Sheep
D003185:Complement C8; D003186:Complement C9; D015938:Complement Membrane Attack Complex; D007457:Iodine Radioisotopes; D011951:Receptors, Complement; D003165:Complement System Proteins