The long term goal of this proposal is to identify the primary molecular defects in erythrocyte membrane skeletons causing certain hereditary hemolytic anemias and employ these mutations to probe the intricacies of normal membrane skeleton architecture. Recently developed biochemical methods will be systematically used to measure specific protein concentrations, binding interactions, and possible structural differences in spectrin and other RBC membrane skeleton proteins. I. Is partial spectrin deficiency the principal structural defect in hereditary spherocytosis (HS), and why is the spectrin deficient? Concentrations of spectrin, ankyrin, band 3 and other proteins will be determined by analysis of SDS-PAGE slabs and by RIAs. Effects probably secondary to spectrin deficiency will be measured including lipid deficiencies, increased osmotic fragility, certain biophysical parameters, and clinical severity. The possibility of progression of spectrin deficiency will be approached with density gradient fractionation and in vitro aging. HS spectrin will be examined for intrinsic defects by 2D peptide fingerprinting, electron microscopy, and specific binding interactions between spectrin chains and associated proteins. Possible roles for newly identified calmodulin binding proteins, tropomyosin, and myosin will be sought by comparative analyses of HS and normal RBCs. II. What are the molecular defects in 6 recessive mouse mutations resulting in spectrin-deficient spherocytosis resembling non-dominant human HS? RBC membranes and proteins will be biochemically analyzed as described for human HS. Investigation of the biosynthesis, assembly and turnover of these proteins will be conducted utilizing immunoprecipitations of in vitro 35S-methionine labeled spectrin, ankyrin and band 3. III. Significance of membrane skeleton linkage with the membrane and possible pathological consequences thereof will be studied in RBC membranes from individuals with defective ankyrin binding sites, individuals with reduced 4.1, and Ca2+ ionophore induced spheroechinocytes with predominantly 2.3 anykyrin. Possible roles for newly identified calmodulin-binding proteins, tropomyosin, and myosin will be investigated in these cells. Band 3 oligomerization and 43K fragment analysis will be conducted on RBCs with reduced ankyrin sites. 4.1 binding will be measured in 4.1 deficient RBCs. The importance of ankyrin 2.3 will be sought.
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