The purposes of this study were to demonstrate the C3 convertase complex, C3b,Bb (EC 126.96.36.199), of the alternative pathway of complement by ultracentrifugation and to determine whether the metal ion required for enzyme formation is present in the active enzyme complex. It has been shown previously that C3b,Bb formed with Ni2+ rather than Mg2+ exhibits enhanced stability. Using sucrose density gradient ultracentrifugation, an enzymatically active C3b, Bb(Ni) complex could be demonstrated which has a sedimentation coefficient of 10.7 S and which is stable in 10 mM EDTA. Upon formation of the enzyme with the radioisotope 63Ni2+, the ultracentrifugal distribution of the metal correlated with that of the enzyme complex. The molar ratio of Ni to C3b,Bb was 1:1. Displacement of Ni by Mg during formation of the enzyme indicated that both metals may bind to the same site in the enzyme. Binding of 63Ni to the catalytic site bearing fragment Bb was significantly stronger than its binding to C3b or to the zymogen, Factor B. It is proposed that there is one metal-binding site in the C3b,Bb enzyme which is not susceptible to chelation by EDTA and which is located in the Bb subunit.
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1983|