COVALENT LINKAGE OF C3 TO PROPERDIN DURING COMPLEMENT ACTIVATION

Activation of the alternative complement pathway of serum produces com plexes of properdin (P) and C3 as measured in a double antibody enzyme -linked immunosorbent assay. When purified from serum, these complexes decrease factor B hemolytic activity in serum and do not restore the alternative pathway hemolytic activity of serum deficient in P. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of activ ated serum containing biotinylated P, followed by blotting to nitrocel lulose and development with streptavidin-alkaline phosphatase revealed a band at 53 kDa for monomeric P and an additional band at 160 kDa. P samples eluted from zymosan and purified from activated serum reveale d a band at 116 kDa for C3 alpha, and 74 kDa for C3 beta, and an addit ional band at 160 kDa when analyzed by SDS-PAGE, Western blotting and development with antibody to C3. The appearance of a 160 kDa band cont aining P and C3 indicates that these proteins are contained in a compl ex formed during activation of the alternative pathway. Activation of a purified reagent mixture containing factors B, D, and H, and I-125-l abeled P or I-125-labeled C3, followed by SDS-PAGE and autoradiography confirmed the presence of a 160-kDa band which disappeared following hydroxylamine treatment of the sample. These data are consistent with a covalent linkage of C3 to P via the C3 alpha chain, producing the 16 0-kDa complex.