Abstract
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.
Highlights
Complement protein C6 has been proposed by othehrsuman C6 as an additional proteolytic enzyme of the completo be aserine protease whoseactivity is obligatory for ment system whose activity was obligatory for complement
The physical association of functionally active C(5,6)".,C6-associated ATIII with C5b-9 is analogous to that observed in stable thrombin is not required for formation of functional ATIII-protease complexes (10,ll)
The comparativedistribution of protein, C6 hemolytic activity, and amidase activity of fractions eluted from the DEAE-Sephacel column (Fig. 2) showedthat a peak of amidase activity was eluted slightly ahead of C6
Summary
Complement protein C6 has been proposed by othehrsuman C6 as an additional proteolytic enzyme of the completo be aserine protease whoseactivity is obligatory for ment system whose activity was obligatory for complement-. We separated theserine mediated target cell lysis. This claim was based on the obprotease (Mr 30,000) activity found associatedwith served inactivation of C6 hemolytic activity by inhibitors of apparently homogeneous preparations of C6 from the serine proteases IPrzPF,' PMSF,andp-APMSF) and hemolytically active C6 protein.Theprotease was characterized as thrombin-likebased on substrate specificity, inhibitor profile, and kinetic studies. The proteolytiacctivity of C6preparations was inhibitable by several inhibitors of serine proteases, the C6 hemolytic activity remained unaffected. - cytolytic activity in the presence of C7, C8, and C9; proteases [7,8],was firmly bound to thecytolytically inactive butno cleavage ofthe a-chain of C5 was observed. The physical association of functionally active C(5,6)".,C6-associated ATIII with C5b-9 is analogous to that observed in stable thrombin is not required for formation of functional ATIII-protease complexes (10,ll).
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