This project will continue our studies on microtubule (MT) protein focusing primarily on structural investigations both within and outside MT. Specifically, we plan to study a) the exchangeable nucleotide binding site with respect to conformational changes occurring during polymerization/depolymerization, and amino acid composition at this site on the Beta subunit; b) the identification and functional characterization of microtubule-associated proteins (MAPs); c) the interaction of MT with other structural platelet proteins e.g. myosin and actin; d) the association of MT with membrane components and e) the characteristics of cold-stable MT. Items c) and d) are continuation projects of previous investigations. The hypothesis which forms the conceptual framework for many of these studies postulates an indirect linkage between membranes and MT, possibly through the interposition of certain MAPs. Binding of platelet activators to receptors that are connected to MT dissociates the latter by changing the number of phosphate groups on polyphosphoinositides. This system sensitizes platelets to the action of aggregating agents. The effect of Ca++ and Ca++ calmodulin on structural aspects of MT and their MAPs will be intensely investigated. Changes in phosphorylation of tubulin and MAPs will be identified and correlated to the state of assembly/disassembly of platelet MT. Cold-stable MT which exist in platelets will be investigated for characteristics that make them temperature-resistant. The methodology for these studies includes one and two dimensional polyacrylamide gel electrophoresis, autofluorography, nonradiative energy transfer measurements, analysis of peptides and amino acids, covalent linking of neighboring proteins by cleavable crosslinking agents and electron microscopic identification of certain proteins by ferritin-conjugated antibodies. All investigations will be performed with microtubule protein isolated from human platelets. These studies are fundamental in nature and broaden our general understanding of the molecular events early in the course of platelet activation. Their significance, however, also extends into practical aspects of platelet physiology, especially in the field of cold (4 C) storage and preservation.
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