In vitro studies on the biosynthesis of rabbit C3: evidence for the synthesis of a nascent single chain precursor C3.

Biosynthesis of C3 in rabbits was studied by examining the incorporation of 14C or 3H-labeled amino acids into C3 immunoprecipitable materials by liver organ cultures and by the in vitro cell-free translation of isolated liver poly A-mRNA in the rabbit reticulocyte system. On SDS polyacrylamide gel electrophoresis, 14C-C3 immunoprecipitated from the extracellular culture medium migrated with an apparent m.w. of 200,000 daltons. In the presence of 2-mercaptoethanol it was found to dissociate into two polypeptide chains, the α chain, m.w. 130,000 and the β chain, m.w. 84,000. On the other hand, 14C-C3 immunoprecipitated from the postmitochondrial supernatant of the homogenized organ explants (intracellular C3) migrated as a single protein on SDS gels even in the presence of reducing agents. Poly A-mRNA isolated from liver by phenol/chloroform extraction and column chromatography on oligo-(dT) cellulose was shown to direct the incorporation of 3H-leucine into TCA-precipitable protein in a concentration and time-dependent manner. Under optimal conditions the addition of exogenous mRNA resulted in a 5- to 6-fold increase in protein synthesis. The immunoprecipitated 3H-C3 migrated as a single peak of m.w. 200,000 under both reducing and nonreducing conditions. These results, taken in conjunction with the results obtained from the liver organ culture experiments, would suggest that C3 is initially synthesized as a single chain precursor protein and later modified to give the two-chain form found in serum.