title: ESR studies of the erythrocyte membrane skeletal protein network: influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates.
abstract: The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.
mesh_terms: D002236:Carbohydrate Conformation; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004910:Erythrocyte Membrane / Q000648:ultrastructure*; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000097:blood*; D011487:Protein Conformation; D013049:Spectrin / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology; D013113:Spin Labels
chemical_list: D006023:Glycoproteins; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008565:Membrane Proteins; D013113:Spin Labels; D013049:Spectrin; D013096:Spermine