Interaction of C3b, B, and D̄ in the Alternative Pathway of Complement Activation

Abstract

The interaction of ZX^d2, an insoluble intermediate of the alternative pathway on zymosan (Z⁵), with factor B and the enzyme D̄ proceeds in a two-step reaction: 1) B binds in the presence of Mg⁺⁺ to ZX^d2 to form the intermediate ZX^d2B, 2) B bound to ZX^d2 is subsequently activated enzymatically by D̄ to yield the complex ZX^d2B̄ which cleaves C3. Evidence was obtained that C3b, which is present on ZX^d2, is required for ZX^d2B̄ formation. Studies of the functional role of C3b for ZX^d2B̄ formation revealed that C3b is involved in the first reaction step, i.e., binding of B to ZX^d2 to yield ZX^d2B. Formation of ZX^d2B is inhibited by pretreatment of ZX^d2 with either anti-C3 Fab or with C3b-INA. Low ionic strength of about 2 mS was found to favor the interaction of the C3b with B. Mg⁺⁺ concentrations from 1 to 31 mM as well as variation of pH in the range from 6.2 to 8.5 did not greatly influence the reaction of B with ZX^d2. For the enzymatic activation of B only C3b on ZX^d2 and factor D̄ are required. This is concluded from the finding that fluid phase C3b is sufficient for the activation of B in the presence of D̄. This does not exclude the fact that other proteins present on ZX^d2 may help to stabilize the intermediate ZX^d2B or the enzymatically active complex ZX^d2B̄, or both of them.