Isolation and functional characterization of the proenzyme form of the catalytic domains of human C1r.
The proenzyme form of C1r catalytic domains was generated by limited proteolysis of native C1r with thermolysin in the presence of 4-nitrophenyl-4'-guanidinobenzoate. The final preparation, isolated by high-pressure gel permeation in the presence of 2 M-NaCl, was 70-75% proenzyme and consisted of a dimeric association of two gamma B domains, each resulting from cleavage of peptide bonds at positions 285 and 286 of C1r. Like native C1r, the isolated domains autoactivated upon incubation at 37 degrees C. Activation was inhibited by 4-nitrophenyl-4'-guanidinobenzoate but was nearly insensitive to di-isopropyl phosphorofluoridate; likewise, compared to pH 7.4, the rate of activation was decreased at pH 5.0, but was not modified at pH 10.0. In contrast, activation of the (gamma B)2 domains was totally insensitive to Ca2+. Activation of the catalytic domains, which was correlated with an irreversible increase of intrinsic fluorescence, comparable with that previously observed with native C1r [Villiers, Arlaud & Colomb (1983) Biochem. J. 215, 369-375], was reversibly inhibited at high ionic strength (2 M-NaCl), presumably through stabilization of a non-activatable conformational state. Detailed comparison of the properties of native C1r and its catalytic domains indicates that the latter contain all the structural elements that are necessary for intramolecular activation, but probably lack a regulatory mechanism associated with the N-terminal alpha beta region of C1r.
Lacroix
M B
MB
Département de Recherches Fondamentales (Unité INSERM 238), Centre d'Etudes Nucléaires de Grenoble, France.
Aude
C A
CA
Arlaud
G J
GJ
Colomb
M G
MG
D002384:Catalysis; D002412:Cations; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003172:Complement C1 / Q000378:metabolism*; D015923:Complement C1r; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D013050:Spectrometry, Fluorescence
D002412:Cations; D003172:Complement C1; D004792:Enzyme Precursors; D003166:Complement Activating Enzymes; D015923:Complement C1r