Activation of Rabbit C3: Studies of the Generation of Cleavage Products in vitro and of Their Metabolism in vivo

Abstract

The cleavage of purified, functionally active rabbit C3 by cobra venom factor and trypsin was analysed by reducing and non-reducing sodium dodecyl sulphate electrophoresis and autoradiography. The specific aim of the study was to compare these reactions to those that occur with human C3. Analysis showed that the pattern of breakdown was very similar to that for the human protein: while the beta-chain remained intact, there was step-wise degradation of the alpha-chain to form C3a, C3b, iC3b and C3c, all of which could be identified by gel analysis. The metabolic behaviour of three of these cleavage products, C3a, C3b and iC3b, was then examined in vivo using dual isotope techniques. Rabbits were studied simultaneously with 131I-C3 and 125I-labelled C3 breakdown products. Analysis of plasma and urine radioactivity for the subsequent 72 h showed that all three breakdown proteins had rapid rates of catabolism in vivo compared to the native molecule. Specifically, 93 and 98% of C3b and iC3b, respectively, were eliminated from the plasma compartment within 10 h of injection. C3a was completely eliminated within 10 h. By comparison, native C3 showed a half-life of 29 +/- 3 h (mean +/- SD) and a fractional catabolic rate of 4.30 +/- 0.75%/h. The data support the use of this species in studies of complement behaviour in models of human immune disease and further clarify the basis for changes in plasma C3 concentration that accompany active immune complex- and antibody-mediated activity, in vivo.