Biochemical characterization of the human complement protein C6. Association with alpha-thrombin-like enzyme and absence of serine protease activity in cytolytically active C6.
Complement protein C6 has been proposed by others to be a serine protease whose activity is obligatory for complement-directed cell lysis. We separated the serine protease (Mr approximately 30,000) activity found associated with apparently homogeneous preparations of C6 from the hemolytically active C6 protein. The protease was characterized as thrombin-like based on substrate specificity, inhibitor profile, and kinetic studies. Although the proteolytic activity of C6 preparations was inhibitable by several inhibitors of serine proteases, the C6 hemolytic activity remained unaffected. Acid-induced (C(5,6)a complex formation between C5 and C6 (protease-free) was demonstrated by ion-exchange fast protein liquid chromatography, reversed-phase high performance liquid chromatography, and reactive cytolytic activity in the presence of C7, C8, and C9; but no cleavage of the alpha-chain of C5 was observed. Diisopropylphosphorofluoridate pretreatment of the components did not affect their ability to generate functionally active (C(5,6)a. Evidently, C6-associated thrombin is not required for formation of functional C(5,6)a. Thus, C6 does not function in the membrane attack pathway of complement as a serine protease. A method for the isolation of homogeneous C6 in the hemolytically fully active form is described. No free sulfhydryl group was detected in C6. The amino acid sequence of 20 amino-terminal residues was determined.
Chakravarti
D N
DN
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Muller-Eberhard
H J
HJ
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003183:Complement C6 / Q000302:isolation & purification / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
D003183:Complement C6; D012697:Serine Endopeptidases; D013917:Thrombin