Three major questions are being addressed in this application: 1. Transmembrane communication in sickle cell anemia red cells.
Our aim i s to identify, at a molecular level, membrane protein interactions which communicate an intracellular event (i.e. hemoglobin S polymerization or aggregation) across the membrane and produce surface alterations leading to cell adherence to vascular endothelium. The experimental approach will include: studies of the associations of polymerized and denatured hemoglobin S with peripheral and integral membrane proteins, comparisons of lateral mobility and clustering of glycophorin A and band 3, and examination of effects of various skeletal pertubations on glycophorin A and band 3 mobility, distribution and adherence of red cells or ghosts to vaccular endothelium and macrophages. 2. Effect of hemoglobin on skeletal protien association. We have recently observed that hemoglobin stabilizes spectrin in the membrane in the tetrametric form. We will characterize the hemoglobin effect on spectrin dimer self association (kinetics, effect on the association constant) the specificity (effect of other proteins, effect of modified or mutant hemoglobins) and the pathophysiologic relevance (studies of the structural integrity of skeletons or ghosts reconstituted with hemoglobin or other proteins). 3. Role of hemoglobin membrane interaction in membrane injury of sickled cells. We will test whether the inhibition of hemoglobin binding to the membrane inhibits the transformation of hemoglobin S red cells into sickled shape and consequently, prevents concommitant alterations in membrane permeability including clacium influx, potassium loss and, ultimately, cell dehydration and red cell-endothelial cell adherence.
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