Covalent linkage of C3 to properdin during complement activation.
Activation of the alternative complement pathway of serum produces complexes 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 activated serum containing biotinylated P, followed by blotting to nitrocellulose 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 revealed a band at 116 kDa for C3 alpha and 74 kDa for C3 beta, and an additional band at 160 kDa when analyzed by SDS-PAGE, Western blotting and development with antibody to C3. The appearance of a 160 kDa band containing P and C3 indicates that these proteins are contained in a complex formed during activation of the alternative pathway. Activation of a purified reagent mixture containing factors B, D, and H, and 125I-labeled P or 125I-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 160-kDa complex.
Whiteman
L Y
LY
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
Purkall
D B
DB
Ruddy
S
S
D003175:Complement C2 / Q000172:deficiency; D003179:Complement C3b / Q000378:metabolism*; D003170:Complement Pathway, Alternative; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006461:Hemolysis; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D011414:Properdin / Q000378:metabolism*
D003175:Complement C2; D006898:Hydroxylamines; D046911:Macromolecular Substances; D011414:Properdin; D019811:Hydroxylamine; D003179:Complement C3b