The long range goal of work in this laboratory is to determine the relationship between protein structure and the overall shape of the red cell. Since the erythrocyte membrane shares many structural elements with other plasma membranes, these relationships should be of general significance for all cells. The particular aims of this proposal are two: 1) To determine the exact location of covalent phosphate or spectrin, and to determine if different phosphorylation sites influence different functions of the molecule; 2) to clarify the effect of Ca on phosphorylation. Ca is known to alter membrane shape, permeability and polypeptide structure. In the work poposed here, the relationship with cell shape will be stressed. The status of the proposed calmodulin dependent kinase will be assessed, and its possible role in membrane phosphorylation determined. The phosphorylated region of spectrin will be cleaved, purified and sequenced, and the location of phosphorylated sites determined. The turnover rate of each of the sites will be measured, both in the intact cell, in membrane skeletons and in the isolated molecule. The phosphorylation state of particular amino acid residues will be determined by partial protease digestion and 2D fingerprinting, and the effect of metabolic depletion, shape changes and possible oligomerization will be studied. Ca is known to change erythrocyte shape and to alter phosphorylation. The underlying mechanism of the Ca effect will be examined, to determine the role of various kinases, phosphatases, or more likely, substrate alteration in the Ca-dependent phosphorylation. The relationship between Ca-induced crenation in the erythrocyte and particular phospho-amino acids in spectrin will be evaluated, using the spectrin sequence data obtained earlier. This work has relevance to hemolytic anemias, in which intra-erythrocytic Ca is often elevated. In addition, the close resemblance between the red cell membrane and other plasma membrane suggests that this work will have relevance to many other cell types.
