title: Purified proenzyme C1r. Some characteristics of its activation and subsequent proteolytic cleavage.
abstract: 1. Upon incubation for 1 h at 37 degrees C, proenzymic C1r was activated by a proteolytic cleavage comparable to that observed in vivo; after reduction and alkylation, two fragments of apparent molecular weights 57 000 and 35 000 were evident on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The activation kinetics were slightly sigmoidal and nearly independent of C1r concentration. They were characterized by a marked thermal dependence (activation energy = 45 kcal/mol). The reaction was inhibited by calcium and p-nitrophenyl-p'-guanidinobenzoate, but poorly sensitive to di-isopropyl phosphorofluoridate. The dependence of the activation rate on pH was unusual; it decreased progressively in the acid range (pH 4.5-6.5) which coincides with the dissociation of the C1r-C1r dimer. Above pH 6.5, the rate increased slightly and showed no clear maximum. These results are consistent with an intramolecular autocatalytic activation mechanism involving the pro-site of each subunit of the C1r-C1r dimer. 2. During a 5 h incubation period at 37 degrees C, C1r underwent two proteolytic cleavages which led to the successive removal of two fragments, alpha (35 000) and beta (7000-11 000) from each subunit, leaving a dimeric molecule of reduced size (Mr = 110 000; s20,w = 6.1 S). The proteolytic process was nearly independent of C1r concentration and characterized by a pH optimum at 8.5-9.0, and a high activation energy (36.8 kcal/mol). Calcium and p-nitrophenyl-p'-guanidinobenzoate, and also di-isopropyl phosphorofluoridate and benzamidine were inhibitors of this reaction. The product, C1r II, retained the original antigenic properties of C1r and a functional active site, but lost the capacity to bind C1s. These results are consistent with an autocatalytic intramolecular proteolysis mediated by the active site of each subunit of the C1r-C1r dimer.
mesh_terms: D003166:Complement Activating Enzymes / Q000378:metabolism*; D003172:Complement C1 / Q000378:metabolism*; D003174:Complement C1 Inactivator Proteins; D015923:Complement C1r; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D011506:Proteins / Q000378:metabolism; D013696:Temperature
chemical_list: D003172:Complement C1; D003174:Complement C1 Inactivator Proteins; D004792:Enzyme Precursors; D011506:Proteins; D003166:Complement Activating Enzymes; D015923:Complement C1r