Dr. Goodman's laboratory has made the first identification of an immunoreactive, structurally related analogue of red blood cell spectrin in various nonerythroid cells (Goodman, Zagon, and Kulikowski, 1981, Proc. Natl. Acad. Sci. 78:7570-7574). Recently, we have identified and purified an immunoreactive analogue of rbc spectrin from a membrane fraction of mouse brain tissue. The purified brain spectrin-like molecule consists of two polypeptides of 240,000 and 235,000 Mr, and on rate zonal sedimentation demonstrates and S20,w = 11, characteristics similar to rbc spectrin tetramer, and brain fodrin. In studies described in this proposal we will ascertain the extent of sequence homology between these three proteins by one dimensional and two dimensional peptide mapping of radioiodinated polypeptides. The structural morphology of the brain spectrin-like molecule will be observed by rotary shadowing and electronmicroscopy, and compared to rbc spectrin tetramer and fodrin. The localization of the spectrin-like molecule within regions of mouse brain tissue (eg. cerebellum) as well as its subcellular localization, will be ascertained by indirect immunofluorescence utilizing an affinity purified anti-mouse -brain spectrin-like protein IgG, and immunoelectronmicroscopy with the ferritin-labelled antibody. The brain spectrin-like molecule is membrane associated, and is a logical candidate to play an essential role in mediating the association of actin with cell membranes. We will therefore study the ability of purified brain spectrin-like protein to bind the erythrocyte membrane attachment proteins, protein 2.1 (syndein) and protein 4.1 in vitro. The ability of purified brain spectrin-like protein to bind F-actin plus/minus protein 4.1 will be studied in solution. If specific binding is demonstrated the location of these binding sites will be visualized by rotary shadowing. We will study the content of the membrane-associated spectrin-like protein in brain tissue from sph/sph mice, which genetically lack erythrocyte spectrin causing profound hereditary spherocytosis and extraordinary membrane instability. If the sph/sph mice are deficient in brain spectrin-like protein, the morphological consequences to the brain tissue will be observed.
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