Interaction between erythrocyte membrane proteins and complement components. II. The Identification and peptide composition of complement components C3 and C4 desorbed from erythrocyte membranes

Abstract

1. (1) Following lysis of sheep erythrocytes with antibody plus human complement, several serum proteins can be desorbed from washed membranes by incubating these in isotonic buffer at 37 °C. We have analyzed these desorbed proteins by isoelectric focusing combined with sodium dodecylsulfate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 2. (2) Crossed immunoelectrophoresis using antisera to human serum yields complex patterns of precipitation arcs, but the use of monospecific anti-C3 and anti-C4 allows us to identify these components. 3. (3) Isoelectric focusing in polyacrylamide followed by sodium dodecylsulfatepolyacrylamide gel electrophoresis at right angels resolves the desorbed serum proteins into three major components with characteristic isoelectric points and electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gel electrophoresis. One component is serum albumin, non-specifically absorbed. Preparative isoelectric focusing followed by crossed immunoelectrophoresis shows that the other two components comprise desorbed C3 and C4 components of complement. Their isoelectric points are 5.1 and 5.7–6.2 respectively. The desorbed C3 components comprise two entities with apparent molecular weights of 160–180 000. Disulfide cleavage with dithiothreitol splits these into two major components of apparent molecular weight 80 000 and 45 000 and one minor component of 30 000. Desorbed C4 also resolves into two closely associated bands of apparent molecular weight 160 000–170 000 in sodium dodecylsulfate gel electrophoresis. Dithiothreitol treatment reduces these to subunits of apparent molecular weight 80 000, 32 000 and 30 000. Moreover, the appearance of one protein with decreased relative mobility is obserbed. 4. (4) These data are discussed in the light of recent findings based on two-dimensional separations of membrane proteins following complement-mediated lysis. They lead us to conclude that after solubilisation of complement-treated membranes in sodium dodecylsulfate, molecular complexes consisting of membrane-bound C3 and membrane proteins persist in solution. These complexes are stabilized by the intramolecular disulfide bonds of C3