The separation of functionally distinct forms of the third component of human complement (C3).
Complement component C3 prepared by the method of Tack & Prahl [(1976) Biochemistry 15, 4513-4521] was found to contain the following trace contaminants: C3b, haemolytically inactive C3 with intact alpha- and beta-chains (C3u) and degraded C3 (apparent mol.wt. 140000) with an intact beta-chain but with a fragmented alpha-chain. The proportion of C3u in the C3 is increased on standing and by freezing and thawing. These contaminants could be separated from each other and from native C3 by chromatography on sulphated Sepharose. They have been characterized by their susceptibility to C3b inactivator in the presence of beta 1H, their ability to be cleaved by C3 convertase and their ability to form alternative-pathway C3 convertase in solution. Incubation of C3b or C3u with beta 1H and C3b inactivator resulted in cleavage of the C3 species; the alpha'-chain of C3b was cleaved to fragments of apparent mol.wts. 67000 and 43000, the alpha-chain of C3u was cleaved to fragments of apparent mol.wt. 75000 and 43000. Native C3 and degraded C3 were unaffected by incubation with beta 1H and C3b inactivator. C3u, unlike C3, was not cleaved to C3b by the classical- or alternative-pathway C3 convertase in solution. When C3b or C3 was incubated with factors B and D, forming C3 convertase, the initial rate of factor-B cleavage was several order of magnitude lower in the presence of C3 than in the presence of C3b. The slow rate observed for C3 could be decreased by preincubation with beta 1H and C3b inactivator or by rechromatography of the C3. The degraded C3 did not support factor-B cleavage by factor D.
D002846:Chromatography, Affinity / Q000379:methods; D003176:Complement C3 / Q000302:isolation & purification*; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003179:Complement C3b / Q000302:isolation & purification; D003180:Complement C3b Inactivator Proteins / Q000378:metabolism; D017242:Complement Factor H; D003170:Complement Pathway, Alternative; D004591:Electrophoresis, Polyacrylamide Gel
C493495:CFH protein, human; D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D003179:Complement C3b; D017242:Complement Factor H; D050577:Complement C3-C5 Convertases