The interaction of C5 with C3b in free solution: a sufficient condition for cleavage by a fluid phase C3/C5 convertase.
We have measured the interactions of C3b with C5 in free solution under conditions that favor detecting weak binding interactions (high C3b and low C5 concentrations and low ionic strength). When a mixture of 125I-C5 (2 X 10(-8) M) and unlabeled C3b (3.8 x 10(-5) M) was ultracentrifuged in a sucrose gradient, virtually all of the C5 sedimented to the position of a 13 to 14S complex. In contrast, a sedimentation rate for C5 of 9S was obtained in the absence of C3b. The ability to bind C5 was observed to be a property of C3b since native C3 was unable to bind C5. It was also found that beta 1H by itself could inhibit the binding of C5 to cell-bound C3b. From inhibition studies, we estimate that the association constant for the C3b-C5 interaction is on the order of 2 x 10(6) M-1 in a low ionic strength buffer (mu = 0.06) and 5-fold weaker at physiologic ionic strength. C5 bound to C3b in free solution was cleaved by nephritic factor-stabilized fluid phase C3bB. C5 activation did not occur on omitting C3b. We conclude that the ability to bind C5 is a property of C3b molecules whether surface bound or in free solution and that when C5 is bound in either fashion, it can be cleaved by a fluid phase C3/C5 convertase.
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003166:Complement Activating Enzymes / Q000276:immunology*; D050577:Complement C3-C5 Convertases / Q000276:immunology*; D003179:Complement C3b / Q000276:immunology*; D003182:Complement C5 / Q000276:immunology*; D011415:Complement Factor B / Q000276:immunology; D003170:Complement Pathway, Alternative; D006801:Humans; D012996:Solutions
D003182:Complement C5; D012996:Solutions; D003179:Complement C3b; D003166:Complement Activating Enzymes; D050577:Complement C3-C5 Convertases; D011415:Complement Factor B