The complement system in rainbow trout (Salmo gairdneri). II. Purification and characterization of the fifth component (C5).

Abstract

The fifth component of rainbow trout complement was purified to homogeneity using a 4-step purification procedure: 1) 5 to 15% polyethylene glycol precipitation; 2) DEAE-Sepharose column chromatography; 3) gel filtration on Sephadex G-200; and 4) CM-Sepharose column chromatography. A 175-fold purification was attained with 20 to 30% yield of C5 antigen and hemolytic activity. The rainbow trout C5 thus purified was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. Rainbow trout C5 was composed of 2 polypeptide chains with m.w. of 133,000 daltons and 86,000 daltons linked by disulfide bonds. The highly purified C5 retained its functional activity. It fully reconstituted the hemolytic titer to C5-depleted rainbow trout serum. Upon activation, this protein was incorporated into a macromolecular complex that closely resembles a membrane attack complex (MAC) of human and guinea pig complement. On the basis of physico-chemical and functional characteristics, we identified this protein as the rainbow trout counterpart of mammalian C5. The identification of C5 in rainbow trout serum strengthened our previous conclusion that the complement system is in a highly developed state already at the phylogenetic level of teleost fish.