Formation and composition of the C3 activating enzyme complex of the properdin system. Sequential assembly of its components on solid-phase trypsin-agarose.
An active C3 cleaving enzyme is generated when properdin factor B (glycine-rich beta-glycoprotein, GBG) is activated (GBG is cleaved) by factor D in the presence of C3b and Mg++. Factor D can be replaced by trypsin in this reaction but even then C3b and Mg++ are required. In the absence of C3b and/or Mg++ trypsin does not generate C3 cleaving activity from GBG although it cleaves GBG and releases its GGG fragment (B) under these conditions as well. Addition of C3b to GGG immediately after its release does not yield a C3 cleaving enzyme. These findings indicate that C3b, GBG and Mg++ interact, and that only in association with C3b can GBG be cleaved in a way that its enzyme activity, residing in a C3b, GGG complex, is expressed. The complex is labile (half-life at 20 degrees C: 9 minutes); Mg++ does not affect its stability nor is it essential for the activity. It was possible to sequentially fix on agarose the essential components of the C3 cleaving enzyme and thus to elucidate the single steps and the order of its formation. C3 was activated and the resulting C3b fragment fixed on agarose by incubating C3 with trypsin covalently bound to the agarose. The agarose-C3b intermediate was capable of binding GBG provided Mg++ was present. GBG could then be cleaved by factor D or trypsin added in solution; the solid-phase-fixed complex obtained had C3 cleaving activity, in the presence as well as absence of Mg++. Omission of any of these steps or components, or changes in the sequence did not give rise to an active enzyme. Mixtures of C3b, GBG and Mg++ have weak C3 cleaving activity by themselves. C3 cleavage in such incubation mixtures proceeds slowly for hours and is not accompanied by cleavage of GBG. There is thus complete analogy between the CVF-dependent and C3b-dependent C3 cleaving systems. C3b and CVF act in the same way, they form a reversible, weakly active C3 cleaving complex with GBG and Mg++, the activity of which is markedly enhanced but becomes subject to decay when GBG is cleaved by trypsin-like enzymes while bound to CVF or C3b.