Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution.
The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38,000 daltons. Human CobINA was found to be a glycoprotein containing at least 10.7% carbohydrate and to have a normal serum concentration of 34 +/- 7 mug/ml (mean +/- 1 SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating beta1H were removed from these proteins with anti-beta1H Sepharose. However, in the presence of highly purified beta1H and C3bINA, both C3bIna, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their structure. The action of C3bINA and beta1H on C3b produced two fragments of the alpha1-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40,000 daltons. The action of C3bINA and beta1H on C4b resulted in cleavage of the alpha'-chain giving rise to the 150,000-dalton C4c and the 49,000-dalton C4d fragments which dissociated without reduction. To produce from C3b the immunochemically defined C3c and C3d, fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.
D000596:Amino Acids / Q000032:analysis; D000888:Antibodies, Anti-Idiotypic; D001798:Blood Proteins / Q000494:pharmacology*; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D003176:Complement C3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism; D003169:Complement Inactivator Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006801:Humans; D007074:Immunoglobulin G; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D012712:Serum Globulins / Q000302:isolation & purification*
D000596:Amino Acids; D000888:Antibodies, Anti-Idiotypic; D001798:Blood Proteins; D002241:Carbohydrates; D003176:Complement C3; D003181:Complement C4; D003169:Complement Inactivator Proteins; D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D010446:Peptide Fragments; D012712:Serum Globulins