Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r.
Fluorescent probes and other methods have been used to investigate the thermal stability of activated C1r and functionally intact fragments isolated from tryptic digests of the protein. This enzyme exhibits two irreversible transitions that differ with respect to their sensitivity to metal ions. The high-temperature transition occurs with a midpoint near 53 degrees C in 0.02 M tris(hydroxymethyl)aminomethane buffer and 0.15 M NaCl, pH 7.4. It is relatively insensitive to Ca2+ and ionic strength and is accompanied by a loss of catalytic activity. The low-temperature transition is most easily observed in the presence of ethylenediaminetetraacetic acid and is completely abolished by 100 microM Ca2+. Its midpoint varies between 26 degrees C at low ionic strength and 40 degrees C in the presence of 0.5 M NaCl. The low-temperature transition results in extensive polymerization of the protein without loss of the esterolytic activity or the ability to react with C1 inhibitor; however, the ability to reconstitute hemolytically active C1 or even bind to C1s in the presence of Ca2+ is destroyed. A highly purified N-terminal fragment generated by tryptic digestion of C1r in the presence of Ca2+ retained its ability to interact with C1s, disrupting the formation of C1s dimers in the presence of Ca2+. In the absence of Ca2+, this fragment displays only a low-temperature transition that is very similar to the one observed with the whole protein and that destroys its ability to bind to C1s. Addition of Ca2+ stabilizes this fragment, shifting the midpoint of its melting transition upward by more than 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Busby
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American Red Cross Biomedical Research and Development, Rockville, Maryland 20855.
Ingham
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KC
D000817:Anilino Naphthalenesulfonates; D002118:Calcium / Q000494:pharmacology*; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003172:Complement C1 / Q000378:metabolism*; D015923:Complement C1r; D003173:Complement C1s / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D003172:Complement C1; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D003166:Complement Activating Enzymes; D015923:Complement C1r; D003173:Complement C1s; D002118:Calcium