Characterization of the human complement (c3b) receptor with a fluid phase C3b dimer.
The interaction of C3b receptor with C3b, the major cleavage product of C3, elicits important biologic functions, such as enhanced phagocytosis and release of cellular enzymes. We determined the binding kinetics and binding isotherm of C3b-receptor interaction by using human cells and fluid phase C3b generated by trypsin cleavage of purified native C3. 125I labeled C3b was separated into 2 molecular species, a dimer and a monomer by column chromatography. We found that dimeric C3b bound to human erythrocyte C3b receptors with an affinity that was more than 25 times that of the monomer. 125I dimeric C3b did not bind to sheep red blood cells, which lack the C3b receptor, nor to trypsinized or 2-mercaptoethanol treated normal human red blood cells, 2 methods for abrogating the immune adherence activity. Binding of 125I fluid phase C3b dimer to the C3b receptor was specific, saturable (about 90 ng of C3b dimer bound maximally per 1 X 10(9) red blood cells), reversible (in the presence of a 100-fold molar excess of unlabeled ligand), and of moderate affinity (Kd about 9.53 nM). The equilibrium binding constants were similar with the various cells tested. Binding was characterized by rapid on and off rates and did not exhibit ligand cooperativity. This specific interaction reached a steady state within 10 to 15 min at 0 degrees C; 50% of specifically bound ligand dissociated from its binding site on human red blood cells in approximately 1 min at 0 degrees C. The density of C3b receptors on human red blood cells, polymorphonuclear leukocytes, monocytes, and B lymphocyte-enriched preparations was 360, 20,000, 30,000, and 21,000 receptors/cell, respectively.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003179:Complement C3b; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D011108:Polymers; D011817:Rabbits; D011951:Receptors, Complement; D012756:Sheep; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D007457:Iodine Radioisotopes; D011108:Polymers; D011951:Receptors, Complement; D003179:Complement C3b; D014357:Trypsin