Studies of the Molecular Mechanisms of C3b Inactivation and a Simplified Assay of β1H and the C3b Inactivator (C3bINA)

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Abstract
A sensitive radio assay was developed to evaluate and quantitate the function of the control proteins important in the cleavage of cell-bound C3b and in the release of the subfragment C3c. In addition, the effects of the control proteins on C3b functional hemolytic activity and on C3b immune adherence reactivity were measured. The cellular complement intermediate EAC43b was prepared with highly purified radiolabeled C3. The effects of incubation with β1H and the C3b inactivator were followed over a wide range of protein concentrations, and the kinetics of the interaction was measured. Incubation of EAC43b* with β1H led to some decrease in immune adherence activity but no loss in C3b functional hemolytic activity and no change in the integrity of the α- or β-chains. Addition of C3b inactivator to the membrane-bound β1H-C3b complex led to a single cleavage of the C3b α-chain without release of the C3c fragment from the cellular site. This led to complete loss of C3b functional hemolytic activity. The α-chain cleavage led to loss but not complete disappearance of immune adherence reactivity. The action of both β1H and the C3b inactivator on cell-bound C3b led to the appearance of a trypsin-sensitive bond within the α-chain. Addition of trypsin led to a second cleavage of the α-chain and the release of the radiolabeled C3c fragment. In kinetic studies the appearance of the trypsin-sensitive bond was shown to parallel the loss of C3b functional hemolytic activity. Kinetic studies demonstrated that both plasma and serum contain all of the ingredients for a C3b-cleaving system. By utilizing this sensitive radio-release assay, the functional titers of C3b inactivator in the serum of 15 normal individuals were found to be 11,310 ± 666 S.E. The mean β1H titer for 12 normal individuals was 20,600 ± 873. This assay can be used to quantitate the level of the trypsin-like enzyme as well. The assay provides a simple method to quantitate and evaluate the function of the C3b control proteins.

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