Studies on the mechanism of immune cytolysis have been greatly facifitated by the use of highly purified complement components. Paradoxically much can be learned about immune cytolysis by studying the reactions of complement components in solutions which are entirely devoid of cells. The experiments described in the present paper were performed to explore the molecular interactions between the first four components of human complement in cell-and antibody-free solution. Use was made of highly purified preparations of the esterase moiety of the first component (C'1), and of the second (C'2), third (C'3), and fourth (C'4) component. It was found that C'2 and C'4 tend to interact in free solution to form a reversible protein-protein complex which is converted into a stable complex by the action of C'I esterase. Formation of the esterase-induced complex results in the generation of an enzyme-like activity which causes the conversion of CP3 to its hemolytically inactive product, Cr3i (1). Evidence will be presented indicating that the complex is formed not only in cell-free solution, but also on the surface of cells and that it fulfills an essential function in cell membrane damage by complement. Materials and MethodsPurified Human Complement Fa~tors.--The esterase moiety of the first component of complement (2) was isolated and kindly provided by Dr. Irwin Lepow, Cleveland, Ohio.The second component was purified from the hydrazine-trested pseudoglobniin fraction of human serum by a three step procedure which will be described in detail elsewhere. 1 Briefly, the first step consists of chromatography on citrboxymethyl-ceUniose using phosphate buffer, pH 6.0, ionic strength 0.05 containing 0.0025 ~ EDTA. The protein is eluted by an increasing * This is publication No. 190 from
In addition to being part of the classical third component of complement, /31c-globulin (1) has been shown to play a key role in the mechanism of immune adherence (2), conglutination, immune conglutination (3), and erythrophagocytosis in vitro (4) and in vivo (5).Since the various functions of/3m-globtflin appear to depend upon attachment of the protein to the erythrocyte surface, a study was carried out with the aim (a) to delineate the mechanism of transfer of file-globulin from the fluid phase to cell membrane receptor sites, and (b) to evaluate quantitatively the functional significance of cell-bound/3it-globulin. This work became feasible through the development of an improved method of isolation of file-globulin (6) and the use of a radioactive label which permitted detection and quantitation of minute amounts of this protein on cell membranes and in free solution.Evidence will be presented indicating (a) that uptake of/3at-globulin by cells is effected by an enzymatic process in which flat-globulin behaves like substrate and the activated second component of complement like an enzyme moiety, and (b) that immune hemolysis and immune adherence necessitate binding of a multiplicity of fl~c-molecules per cell.Work in this laboratory (6, 7), and in others (8-11), has revealed that the classical third component of complement consists of a number of different serum factors and that/3~c-globulin represents only one of the factors of this group. According to a recently proposed nomenclature (7, 12), these factors are called components three (C'3), five (C'5), six (C'6), etc., C'3 being synonymous with tic-globulin and no longer with the classical third component. Henceforth, the term C'3 will be used exclusively to denote file-globulin and its hemolytic activity.
Normal IgG and myeloma proteins of the IgG1, 2, 3,and 4 subclasses were mixed with human Clq and studied in the analytical ultracentrifuge for complex formation. Binding of IgG to Clq is apparent both from the enlargement of area and from the increase in sedimentation rate of the well-separated schlieren peak of Clq. The accurate determination of binding parameters requires that sedimentation rates be corrected for hydrodynamic interaction, and area measurements corrected for the Johnston-Ogston effect. At the highest immunoglobulin concentrations employed in these studies more than ten IgG molecules are bound to each Clq. If we assume that the number of binding sites must be an integral multiple of 6, then the data best support a 12 binding site model, although an 18 site model cannot be rule out. Myeloma IgG proteins of all subclasses bind to Clq, with affinities decreasing in the order G3 greater than G1 greater than G2 greater than G4. No binding of IgA to Clq could be detected.
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