Soluble C3 proconvertase and convertase of the classical pathway of human complement. Conditions of stabilization in vitro.
Soluble classical-pathway C3 convertase and proconvertase were prepared from purified C4b-C2ox complex in the presence of Ni2+; the two complexes, stable for at least 15 h at 4 degrees C, were isolated by sucrose-density-gradient ultracentrifugation. The C3 convertase alone was able to cleave C3, and its decay was accelerated in the presence of C4-binding protein. The individual roles of Ni2+ and I2 treatment of C2 in the stabilization of the complexes seemed to be different and additive. 63Ni2+ binding coupled to h.p.l.c. analysis showed that 63Ni2+ bound only to the C2ox proteolytic fragment a (1 mol/mol) with a Kd of 26 microM. Competition studies between Ni2+ and Mg2+ indicated that only half of the Ni2+ bound to the C3 convertase was removed by Mg2+, whereas, in the same conditions, Ni2+ bound to C2ox proteolytic fragment a was not displaced, suggesting the presence of two sets of sites on the convertase. EDTA prevented the formation of both C3 convertase and proconvertase; EDTA had no effect on the preformed C3 convertase, whereas it dissociated the preformed proconvertase.
D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003166:Complement Activating Enzymes / Q000378:metabolism*; D050577:Complement C3-C5 Convertases / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004792:Enzyme Precursors; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011506:Proteins / Q000378:metabolism
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011506:Proteins; D009532:Nickel; D004492:Edetic Acid; D003166:Complement Activating Enzymes; D050577:Complement C3-C5 Convertases; D008274:Magnesium