Isolation of the fifth component of the bovine complement system.
Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.
Aston
W P
WP
Department of Microbiology and Immunology, Queen's University, Kingston, Ont., Canada.
Mhatre
A
A
Macrae
J
J
D000818:Animals; D002417:Cattle / Q000276:immunology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003176:Complement C3 / Q000032:analysis; D003180:Complement C3b Inactivator Proteins / Q000032:analysis; D003182:Complement C5 / Q000302:isolation & purification*; D017242:Complement Factor H; D015941:Complement Hemolytic Activity Assay; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight
D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D003182:Complement C5; D017242:Complement Factor H