Purification of proenzymic and activated human C1s free ofC1r. Effect of calcium and ionic strength on activated C1s.
1. A rapid method for the purification of the proenzymic and activated forms of C1s is presented. In the case of proenzymic C1s, di-isopropyl phosphorofluoridate (0.5--5 mM) is added at all stages of the purification procedure, which includes euglobulin precipation followed by DEAE-cellulose chromatography and affinity chromatography on anti-C1r IgG-Sepharose 6B. The final step completely removes contaminant traces of C1r and/or C1r, ensuring that the final preparation of C1s is stable in the proenzyme form and suitable for activation studies. 2. The apparent molecular weight of C1s and C1s determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis is 85 000 +/- 2000. Reduction followed by alkylation of C1s gives two fragments of apparent molecular weights 57 000 and 28 000. Results of N-terminal amino acid determination and labelling with di-iso[3H]propyl phosphorofluoridate are consistent with previous reports. 3. The influence of calcium and ionic strength on the structure and activity of C1s has been investigated. Calcium leads to a shift of the sedimentation coefficient from 4.3 to 5.6 S, whereas variation in ionic strength has no effect on this parameter. The thermal inactivation curve is profoundly modified both by calcium and ionic strength. In contrast, the esterase activity is only slightly influenced as judged from the absence of gross modification of Km and V.
D002118:Calcium / Q000494:pharmacology*; D003166:Complement Activating Enzymes; D003172:Complement C1 / Q000302:isolation & purification*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008722:Methods; D009994:Osmolar Concentration
D003172:Complement C1; D004792:Enzyme Precursors; D003166:Complement Activating Enzymes; D002118:Calcium