PATHOPHYSIOLOGY OF RED CELL MEMBRANE DISEASES

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This proposal is primarily concerned with the structure and function of the membrane skeleton in certain inherited red cell membrane disorders. It is divided into 4 parts: First, we will characterize interactions of the 3 major skeletal proteins (spectrin, actin and 4.1) with each other in the normal red cell. We will measure the stoichiometry and affinity of these interactions, localize the respective binding domains with a new photoaffinity reagent, and determine the physical properties and self-association of protein 4.1. Second, we will identify new membrane skeletal defects by systematically analyzing membrane protein composition, membrane phosphorylation, and each of the skeletal protein interactions using established techniques. Third, we will characterize 3 newly discovered forms of hereditary spherocytosis (HS). In type I HS (deficiency of spectrin) we will focus on the effects of the spectrin deficit on phospholipid asymmetry and we will compare the effects of reconstitution with phosphorylated and dephosphorylated spectrin on membrane stability and integral protein mobility. In type II HS (defect in the 4.1 binding site of spectrin) we will isolate and characterize 2 thiol-containing tryptic peptides that are missing in the defective spectrin and locate these peptides in relation to the 4.1 binding site. We will also study the cellular consequences of this defect, particularly the mechanism of membrane surface loss. In type III HS (unstable spectrin binding site) we will see if the spectrin binding defect is due to an unstable ankyrin and attempt to identify the cause of the instability. Fourth we will analyze skeletal protein interactions in stored blood, focusing on a defect in the actin binding site of spectrin that develops early in storage and is corrected by reduction. We will compare the development of this defect to changes in the morphology, surface area, and metabolism of stored cells; analyze thiol groups in the abnormal spectrin; and determine whether the defect and its cellular consequences can be prevented by storage of blood under reducing conditions.