Isolation and functional characterization of the proenzyme form of the catalytic domains of human C1r

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Abstract
The proenzyme form of Clr catalytic domains was generated by limited proteolysis of native Clr 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 Clr. Like native Clr, the isolated domains autoactivated upon incubation at 37.degree. 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 Clr [Villiers, Arlaud and 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 Clr 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 Clr.