Our aim is to study human RBC membrane function in health, senescence, liquid storage and disease. Our approach is to focus on an analysis of reversible and irreversible shape changes in RBC and their ghosts basing experimental designs on the working hypothesis that proteins, lipids, and enzymes are assembled into operational domains within the membrane, and that these assemblies (when modified by smaller molecules like Ca++ or calmodulin to produce both short and long range effects) actually carry our key membrane functions. Specifically 4 related studies are proposed. Analysis of endocytosis in RBC and ghosts provides an opportunity for studying RBC stomatocytosis, invagination, constriction, and fusion along with the fusion role of the membrane cytoskeleton and the lipid bilayer in these events. In addition to forming endocytic vacuoles, BBC membranes can be induced to produce exocytic vesicles, inside-out vesicles, and sheared microvesicles, each of which can be isolated and studied. An understanding of the components that are included and excluded from these subdivided membranes should provide information about membrane assemblies and their alterations during induced budding, internalization, or fragmentation. The Ca++ content of RBCs is meticulously controlled and it is presumed that Ca++ (alone or in concert with calmodulin) modulates important membrane functions. A clue to understanding the role of Ca++ will come from identifying its membrane Ca++ binding sites, which is a project we propose to undertake. Actin is present in the red cell and we have identified a membrane actin activated ATPase which is generally taken to be a hallmark of myosin. We plan to study the effects of CA++ and calmodulin on this activity, and to identify it both by biochemical isolation and immunologic means in order to determine if the red cell has a myosin which would presumably have contractile properties. Using the methods and hypotheses developed in the course of these studies, we plan to analyze the red cell membrane storage lesion and several hemolytic disorders where there is clear or presumptive evidence of membrane abnormality. Our purpose would be to determine how, in these hemolytic disorders, the protein abnormalities affect the membrane function and produce the observed pathophysiology.
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