The proposed research is to study the mechanism of polymerization and gelation of red cell membrane proteins. One of the main goals of this proposal is to develop, in conjunction with the available structure model, the energy map of the cytoskeleton. This work is based on the model which postulates that the cytoskeletal protein form a two-dimensional network of junctions formed by spectrin, Band 4.1 and short oligomers of actin. The proposed kinetic studies, as demonstrated by our pilot experiments, will take advantage of the fluorescence resonance energy transfer between specifically and selectively labeled pairs of energy donors and acceptors conjugated with cytoskeletal proteins. The studies are designed to establish: a. The kinetics of reaction of Band 4.1 with spectrin. b. The effect of the spectrin-Band 4.1 complex on actin oligomerization followed by the increase of fluorescence of pyrene conjugated G-actin. c. The rate limiting step in the assembly of red cell cytoskeleton. d. The magnitude and type of the energy involved in the above interactions. This will be accomplished by microcalorimetric determination of the enthalpy of interactions and by light scattering and osmometric determination of dissociation and association with constants from the apparent molecular weight measurements.
|Podgorski, A; Elbaum, D (1985) Properties of red cell membrane proteins: mechanism of spectrin and band 4.1 interaction. Biochemistry 24:7871-6|