Characterization of the initial C3 convertase of the alternative pathway of human complement.
C3(H2O),Bb, the initial C3 convertase of the alternative complement pathway, was demonstrated to be a metal-containing protein complex by sucrose density gradient ultracentrifugation. Demonstration of this labile enzyme became possible by increasing its half-life with nickel instead of magnesium ions used for enzyme formation. The enzyme was generated from C3, the internal thioester bond of which was hydrolyzed (C3(H2O)), and from 125I-Factor B and Factor D. The sedimentation coefficient of the enzyme complex was 10.7S. By using 63Ni for enzyme formation, the metal ion was detected in the enzyme complex after ultracentrifugation in the presence of 10 mM EDTA. The stoichiometry of the constituents in the C3(H2O),Bb(Ni) complex was 1:1:1. To verify that C3 is incorporated into the enzyme complex in the form of C3(H2O), the enzyme complex was adsorbed to anti-Factor B-Sepharose and subjected to decay-dissociation. Examination of the subsequently eluted protein by SDS gel electrophoresis under reducing conditions demonstrated the presence of an intact C3 alpha-chain. This work provides further evidence that a C3 convertase can be generated from noncleaved C3 that is modified at the thioester site. With the use of a fluorometric assay, the activity (kcat/Km) and the half-life of the initial C3 convertase were determined and compared to those of C3b,Bb.
D055598:Chemical Phenomena; D002621:Chemistry; D003166:Complement Activating Enzymes / Q000378:metabolism; D003167:Complement Activation / Q000187:drug effects; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003170:Complement Pathway, Alternative / Q000187:drug effects; D004355:Drug Stability; D006461:Hemolysis; D006801:Humans; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D008744:Methylamines / Q000378:metabolism; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology; D011868:Radioisotopes / Q000378:metabolism
D008744:Methylamines; D011868:Radioisotopes; D009532:Nickel; D003166:Complement Activating Enzymes; D050577:Complement C3-C5 Convertases; D008274:Magnesium