Interaction of beta1H globulin with cell-bound C3b: quantitative analysis of binding and influence of alternative pathway components on binding.
Purified beta1H globulin (beta1H) was shown to bind to C3b coated cells by both immunofluorescent and radioactive tracer techniques. With EAC43, the amount of beta1H bound was directly proportional to the amount of C3 used to prepare the cells; EA, EAC14 and EAC14oxy2 bound very small amounts of beta1H. The C3b binding site on beta1H was labile in that not all of the purified 125I-beta1H was capable of binding to C3b, even when an excess of cell-bound C3b was present. Scatchard analysis of binding of beta1H to C3b-coated cells indicated an equilibrium constant of 10(9) L/M. Deviations from linearity were regularly found on Scatchard analyses. This was consistent with the hypothesis that the beta1H binding sites exhibit negative cooperativity in that as more sites become occupied, it becomes more difficult to fill the remaining sites. The stoichiometry of the reaction between C3b and beta1H was examined using EAC14oxy23 prepared with 131I-C3 and beta1H labeled with 125I. Between 0.5--0.8 beta1H molecules were bound per C3b molecule. Other alternative pathway components influenced the binding of 125I-beta1H to cell bound C3b. Both C3b and native C3 inhibited binding of labeled beta1H at an efficiency approximately 1/1,000 that of unlabeled beta1H. Factor B inhibited binding with 1/280 the efficiency of unlabeled beta1H. Properdin caused a dose-dependent increase in the binding of beta1H; this enhancement was abrogated if B was also present in the reaction mixture. Scatchard analysis indicated that the enhancement of beta1H binding by P resulted in an increased number of available binding sites rather than an increase in the affinity of binding.
D000818:Animals; D001665:Binding Sites; D003176:Complement C3 / Q000378:metabolism*; D003180:Complement C3b Inactivator Proteins / Q000378:metabolism*; D011415:Complement Factor B / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D011414:Properdin / Q000494:pharmacology; D012756:Sheep
D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D011414:Properdin; D011415:Complement Factor B