The Incorporation of C3 Nephritic Factor (C3NeF) into a Stabilized C3 Convertase, C3bBb(C3NeF), and its Release after Decay of Convertase Function

Abstract

Abstract The stabilized fluid phase C3 convertase formed during the interaction of C3NeF, B, D̄, and C3 and isolated as a 10S complex by sucrose density gradient ultracentrifugation contains C3NeF. The C3NeF incorporated into the 10S complex cannot be detected by hemolytic assay until it is released during the decay of convertase function. The total recovery of the C3NeF present in the original reaction mixture from the 7S and 10S fractions indicates that the stabilizing action of C3NeF on the fluid phase convertase is not associated with an appreciable augmentation or decline in function. Furthermore, the C3NeF recovered after decay of the fluid phase convertase is identical to the starting material in its stabilizing action on erythrocyte-bound C3bBb, its molecular weight of 160,000 daltons, and its isoelectric point of pH 8.6 to 8.7. The incorporation of partially purified C3NeF into the fluid phase amplification convertase with release upon decay of the convertase, followed by removal of the contaminating complement proteins, yielded more than a 100-fold further purification. Thus, the affinity of C3NeF for the amplification convertase afforded a unique method for removing physicochemically similar contaminating proteins. Purified C3NeF, radiolabeled without decrement in functional activity, was incorporated in a 1:1 molar ratio with radiolabeled B into the 10S complex, thereby establishing a molecular composition of the stabilized convertase C3bBb-(C3NeF) of 1:1:1.