A reexamination of the role of magnesium in the human alternative pathway of complement.
The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.
D002499:Centrifugation, Density Gradient; D003167:Complement Activation; D050577:Complement C3-C5 Convertases / Q000096:biosynthesis; D003179:Complement C3b / Q000378:metabolism; D011415:Complement Factor B / Q000378:metabolism; D011416:Complement Factor D / Q000494:pharmacology; D003170:Complement Pathway, Alternative; D004591:Electrophoresis, Polyacrylamide Gel; D006461:Hemolysis; D006801:Humans; D008274:Magnesium / Q000378:metabolism / Q000502:physiology*
D003179:Complement C3b; D050577:Complement C3-C5 Convertases; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B; D008274:Magnesium