SULFANILIC ACID DIAZONIUM SALT - A LABEL FOR OUTSIDE OF HUMAN ERYTHROCYTE MEMBRANE

1. 1. The diazonium salt of [35S]sulfanilic acid can be used as a label for outer components of the human erythrocyte membrane; the reagent does not penetrate intact cells. 2. 2. Modified cells become permeable to Na+ and K+ but not to water-soluble nonelectrolytes. They eventually lyse in isotonic buffer. 3. 3. About 20% of the label bound to intact cells can be recovered in an ethanol-ether membrane extract. Phospholipase D (cabbage) changes the way in which it partitions between ether and water. 4. 4. If the residue left after ethanol-ether extraction is dissolved in 3% sodium dodecyl sulfate and sized by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate, a complex but reproducible pattern is observed on staining with Coomassie brilliant blue. The most intensely labeled material has a molecular weight of about 140 000. Some peaks are free of label. 5. 5. Similar patterns are obtained if intact membranes are dissolved in 3% sodium dodecyl sulfate; sodium dodecyl sulfate dissociates the protein and lipid as effectively as ethanol-ether. 6. 6. If the residue left after ethanol-ether extraction is exposed first to 0.8 M NaCl, a medium in which it is largely insoluble, much of the protein shifts from high to low molecular weight. The most intensely labeled material does not. The membrane contains protein complexes which can be dissociated by sodium dodecyl sulfate after exposure to salt. The dissociation is less extensive when intact membranes are exposed to salt. © 1969.