On the mechanism of cell membrane damage by complement: evidence on insertion of polypeptide chains from C8 and C9 into the lipid bilayer of erythrocytes.
The preceding paper (Hammer, C.H., A. Nicholson, and M. M. Mayer, 1975, Proc. Natl. Acad. Sci., 72:5076) presented evidence on insertion of polypeptide chains from the C5b and C7 subunits of C5b, 6, 7 complex into the phospholipid bilayer of erythrocyte membranes. In the present study, EAC1-8 and EAC1-9 (sheep erythrocytes carrying rabbit antibody and complement proteins C1 through C8 or C9, respectively), prepared with either 125I-C8 or 125I-C9, were incubated with trypsin or chymotrypsin and the release of 125I was measured. Only 9 to 19% of the specifically bound radioactivity was released. In addition, elution experiments were performed with 0.02 M EDTA-1.0 M NaCl. This solution did not elute C9 from EAC1-9. By contrast cellbound C9 was recovered from erythrocyte membranes with sodium dodecyl sulfate (SDS). Thus, enzymatic stripping and elution experiments indicate that cellbound C9 behaves like an integral membrane protein, presumably due to insertion into the lipid bilayer. EAC1-9 membranes that had been subjected to extended digestion with trypsin or chymotrypsin were extracted with SDS to recover the enzyme-resistant part of the C9 molecule from the membrane. Even though this domain of C9 carried 90% of the radioiodine associated with native C9, its m.w. was found to be only 18,000 daltons by analysis on SDS-PAGE. This represents one-quarter of the native C9 molecule.
D000818:Animals; D002793:Cholic Acids / Q000494:pharmacology; D002918:Chymotrypsin / Q000494:pharmacology; D003185:Complement C8 / Q000378:metabolism*; D003186:Complement C9 / Q000378:metabolism*; D003165:Complement System Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000276:immunology*; D004912:Erythrocytes / Q000276:immunology*; D006168:Guinea Pigs; D006801:Humans; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D002793:Cholic Acids; D003185:Complement C8; D003186:Complement C9; D007457:Iodine Radioisotopes; D010455:Peptides; D010743:Phospholipids; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D003165:Complement System Proteins; D002918:Chymotrypsin; D014357:Trypsin