Our object is to study structure-function aspects of the adult human red cell membrane with the aim of using the formulations derived to improve our understanding of the pathophysiology of red cell disorders and of the biology of membranes in general. We propose three lines of investigation to probe structure- function relatiohship at three different levels; that of circulating abnormal RBC as exemplified by the thalassemias; that of a cell biological problem exemplified by shape control in red cells and ghosts; and at the molecular level by investigating the role of the contractile proteins of the red cell and its membrane. (1) In the severe thalassemias unbalanced globin chain synthesis leads to increased RBC and ghost rigidity and abnormalities of the membrane cytoskeleton marked by increases in globin and variations in residual protein 3. The severe alpha thalassemias (Hb H diseaes) have a membrane lesion that leads to hyperstability while in the severe beta thalassemias the ghosts are unstable and fragment easily. We propose to explore the cause of the RBC and membrane deformation and stability abnormalities in each, based on the hypothesis that either bulk binding of globin chain aggregates and/or oxidations mediated by hemichromes in these aggregates causes the membrane lesions that account for the varying pathophysiology of thalassemias. (2) With our recent discovery of myosin light chain kinase, the red cell now has a complete contractile system. We propose to evaluate the possible role of these contractile system in the control of normal and abnormal RBC shape changes with an alternative hypothesis that if the proteins do not function as a contractile system they may subserve a structural role - i.e. myosin may serve as a highly controllable third link between cytoskeletal and integral proteins. (3) Our studies on the control of RBC shape in vitro indicate that the bilayer couple hypothesis cannot easily explain why amphipath induced stomatocytosis, but not echinocytosis, is blocked by vanadate, an inhibitor of phsophohydrolytic reactions. Other observations indicate that in extreme RBC shape changes, like spheroechinoytosis and spherostomatocytossi there is movement of both cytoskeletal proteins and of the intramembrane particles that are composed of the integral transmembrane proteins. We and others have also begun to show that the membrane properties that control elliptical deformation are different than the properties that control the inward and outward membrane bulging that is part of stomatocytosis and echinocytosis. Therefore we propose studies based on a syncretist hypothesis that the full range of RBC shape changes requires the variable interaction of integral proteins and associated phospholipid bilayer with the cytoskeleton, modified by Mg-ATP mediated phosphohydrolytic reactions which may serve to maintain the assymetry of the bilayer.
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