Effect of Reducing Agents on the Structure and the Function of the Third Component of Guinea Pig Complement (C3)

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Abstract
Reduction of guinea pig complement C3 with dithiothreitol or mercaptoethanol at the concentrations of 2.5 mM and higher and the subsequent alkylation of the reduced C3 resulted in the loss of C3 reactivity in immune adherence and immune hemolysis. Treatment of C3 with l-cysteine or alkylation with iodoacetamide alone did not exhibit any detectable inhibitory effect on C3 reactivity up to their concentration of 125 mM. The reactivity of C3, which was treated with dithiothreitol or mercaptoethanol but without subsequent alkylation with iodoacetamide, was shown to recover to almost the original level when reducing agents were removed by dialysis. In contrast to the inhibitory action of reducing agents on the reactivity of C3 in the fluid phase, reduction and alkylation of the cell-bound C3 in the state of either EAC1423, or EAC423 or EAC43 cells did not show any detectable adverse effect on the immune adherence reactivity. The hemolytic reactivity of thus treated hemolytic intermediate cells was significantly affected by treatment with dithiothreitol at the concentrations as low as 2 mM, as already reported. The tests for agglutinability with anti-GPC3 of EAC142 cells reacted with reduced and alkylated C3 revealed the almost complete loss of the capacity of C3 to fix on the surface of EAC142 cells. Therefore, it was suggested that the loss of immune adherence reactivity by reduction and alkylation is a secondary phenomenon due to the failure of specific binding of reduced and alkylated C3 with the surface of EAC142 cells. This hypothesis was supported by the observation that immune adherence reaction between human erythrocytes and tanned sheep erythrocytes to which reduced and alkylated C3 was nonspecifically coupled was not inhibited but was significantly enhanced. This observation suggests that the region of C3 molecules necessary for immune adherence is distinct from the region for specific binding and a kind of activation of the ‘immune adherence region’ of C3 molecules was achieved by reduction and alkylation. Electrophoretic analysis of reduced and alkylated C3 on 5% polyacrylamide gel revealed the occurrence of the split products of C3 with lower molecular weights some of which appeared to be comparable to the fragments of C3 obtained by enzymatic digestion.

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