Characterization of the activation of the human C1r complement molecule.
The proenzyme form of C1r was isolated by sequential chromatography from the euglobulin fraction of human serum on DEAE-Sepharose 6B-CL, CM-Sepharose 6B-CL and Sepharose S-300-CL. This C1r had the tendency to spontaneously activate within 60-90 min of incubation at 37 degrees C in presence of EDTA and more slowly in the presence of Ca2+. The spontaneous activation of C1r was found to be a bimolecular process and could be completely inhibited by DFP in the pH range 6-9 and in the presence of Ca2+ without affecting the hemolytic C1r activity. [14C]DFP bound to trace proteins in the 60-90 kD range, but not to C1r proenzyme. The spontaneous activation of C1r was diminished in the presence of EDTA by DFP, but could not be completely suppressed. EDTA acts by removing Ca2+ from C1r, thereby changing the conformation of the protein and causing an increased digestibility of the C1r H-chain. At temperatures above 0-4 degrees C this influence destroyed the ability of C1r proenzyme and enzyme to form macromolecular C1 and thereby abolished its hemolytic activity. We conclude from these results that the spontaneous C1r activation in the pH range 6-9 and in the presence of Ca2+ is due to contaminant proteases. C1r activated also spontaneously at higher pH values between pH 9 and 13.2, but the spontaneous activation ceased abruptly at pH 13.4. An intramolecular process of activation cannot be excluded at these high pH values. It is, however, not clear, whether this activation is a suitable model for the C1r activation in the C1 molecule, because the hemolytic activity of C1r was substantially diminished under the high pH conditions.
D003166:Complement Activating Enzymes / Q000276:immunology*; D003167:Complement Activation; D015922:Complement C1q; D015923:Complement C1r; D003173:Complement C1s; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006461:Hemolysis; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D012697:Serine Endopeptidases
D007531:Isoflurophate; D015922:Complement C1q; D004492:Edetic Acid; D003166:Complement Activating Enzymes; D010450:Endopeptidases; D012697:Serine Endopeptidases; D015923:Complement C1r; D003173:Complement C1s