Cross linking of erythrocyte membrane proteins and phospholipids by chemical probes.
Membrane proteins and phosphatidylethanolamine (PE) in intact red cells and red cell ghosts were reacted with cross-linking probes, which differed in hydrophobicity and charge. Methylacetimidate (MA) cross-links more spectrin bands 1 and 2 in cells than in ghosts. Band 4.1 reacts readily with MA in both cells and ghosts, whereas band 3 and 5 undergo very little linking. However, band 3 and most membrane proteins except band 5 are extensively cross linked by difluorodinitrobenzene (DFDNB). Band 5 reacts with DFDNB more in ghosts than in cells. Isethionyl acetimidate (IA), a nonpenetrating probe, gives very little cross linking of membrane proteins in intact cells, whereas it extensively cross links proteins in ghosts in a similar fashion as does MA. MA reacts extensively with PE in both cells and ghosts. Depending on the concentration of MA, between 47 and 60% of the PE is cross linked to itself. At 20 mM MA, 94% of PE reacts in intact cells, whereas at 20 mM IA only 19% of PE reacts. Furthermore, 66% of the PE that reacts with IA is cross linked to itself. These studies suggest that hydrophilic imidoesters cross link peripheral membrane proteins such as bands 1, 2, and 4.1, but have restricted access to integral proteins such as band 3. However, the hydrophobic probe extensively cross links both integral and peripheral proteins in intact cells except band 5. These data suggest that rearrangement of some membrane proteins occurs when ghosts are prepared.
D055598:Chemical Phenomena; D002621:Chemistry; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000097:blood*; D010743:Phospholipids / Q000097:blood*; D013696:Temperature
D008565:Membrane Proteins; D010743:Phospholipids