After osmotic lysis, erythrocyte membranes are able to recover impermeability in certain conditions. Many of the factors that induce resealing have been determined. It is therefore, worthwhile to undertake a study of the structural rearrangements that occur in the membrane in going from the permeable to the impermeable state. A simple hypothesis is proposed, based on the observation of distinct permeability barriers for cations and macromolecules, which is capable of experimental testing. The macromolecule barrier is proposed to be a function of an intact lipid bilayer, while cation resealing should require protein-lipid or protein-protein reorganization. This will be tested by observing the effect of compounds that alter lipid fluidity on resealing; by chemical cross-linking studies on the topological orientation of protein-lipid and protein-protein complexes in the membrane; and by assaying the influence of site-specific labelling modifications such as enzymic digestion, specific for one class of membrane component or individual components, on the recovery of the permeability barriers. Emphasis will be placed on the role of (Na,K)ATPase in this process. Many of the same factors that influence resealing rates alter the shape of erythrocytes as well, implying that some membrane components participate in both functions. A study of the shape changes in erythrocyte ghosts will therefore be undertaken, with an emphasis on the effects brought about by the procedures used in studies of resealing. Erythrocyte ghosts will be used; these have the advantage that osmotic effects can be distinguished from intrinsic membrane phenomena, since ghosts can be prepared which are leaky to cations, but can nevertheless exhibit shape changes in response to various conditions. Finally, some effects of intracellular contents on the membrane will be assessed. In the process of resealing, ghosts will trap exogenous molecules. In particular, normal membranes, free of hemoglobin A, can incorporate hemoglobin S; this model of the sickle cell will be used to study the events that lead to the formation of membrane - damaged irreversibly-sickled cells.
NIH ExPorter
NIH ExPorter