Active Disassembly of the First Complement Component, C1̄, by C1̄ Inactivator

Abstract

Abstract In order to clarify its mechanism of disassembly, the first component of human complement, C1, was reconstituted from C1q, 125I-C1r, and 131I-C1s. This radiolabeled C1 bound tightly to aggregated IgG covalently linked to Sepharose 4B. The subsequent addition of C1̄ inactivator (C1̄-In) led to the rapid release of equimolar quantitites of 125I-C1r̅ and 131I-C1s̅ from the agg IgG. SDS-PAGE analyses of such eluates showed that one C1̄-In molecule was associated with each C1r̅ polypeptide chain and another C1̄-In molecule was bound to each C1s̅ polypeptide chain. The released C1r̅, C1s̅, and C1̄-In co-sedimented in sucrose density gradients with a rate of 9S and monospecific anti-C1s̅ pelleted all three proteins. Therefore, C1r̅, C1s̅, and C1̄-In, in a molar ratio of 1:1:2, respectively, are released from C1̄ as a complex. An analogous 9S complex containing C1r̅, C1s̅, and C1̄-In was generated in normal human serum after incubation with activators of the classical complement pathway. The C1r̅C1s̅(C1̄-In) complexes generated in both serum and the purified system were stable in the presence of EDTA. A diffusion coefficient of 2.3 × 10-7 cm2/sec was determined for the C1r̅C1s̅(C1̄-In) complex from its behavior on gel filtration. An m.w. of 330,000 was then calculated from its sedimentation and diffusion coefficients. This m.w. is consistent with the value of 382,000 for a molecule of composition C1r̅C1s̅(C1̄-In)2 obtained by summing the weights of the subunits. These results indicate that C1-In efficiently disassembles C1̄, thereby releasing two C1r̅C1s̅(C1̄-In)2 complexes per C1̄ molecule.