This project is divided into four sections, all of which share a common focus on the role of calmodulin and calcium as regulators of vesicle functions present in nerve endings and of the structural protein molecules around vesicles that participate in the attachment and/or fusion of these organelles with the plasma membrane. The first series of studies focuses on the molecular interactions of clathrin's polypeptides with its associated proteins (CAPs), with the vesicle membrane and with calmodulin in conferring calcium sensitivity to these interactions. For these studies we use affinity-binding chromatography and chemical cross-linkers between amino acid residues of bound proteins. The second set of studies concerns the process of phosphorylation in coated and synaptic vesicles. The identity of the phosphorylated proteins and of the protein kinases found in synaptic and coated vesicles will be determined by their calcium-calmodulin activation patterns, autoradiography of labeled polypeptides resolved after one- or two-dimensional (gel) electrophoresis, and comparison of kinase properties after separation from their corresponding vesicle population. The third set of studies will focus on the calcium/calmodulin-dependent phospholipase A2 activity in both types of vesicles and in synaptosomal membrane preparations. Enzymatic activity will be measured using specifically labeled precursors and the products formed, separated and identified by thin-layer chromatography and/or high-performance liquid chromatography. These enzymes appear to destabilize vesicle membrane, facilitating vesicle aggregation and possibly fusion of vesicles, phenomena that will be examined by light scattering and electron microscopy. The final section of the project will address the separation of these proteins, preparation of specific polyvalent and monoclonal antibodies, and their subcellular localization in brain tissue and cultured cells. We believe that deficient innervation leads to inefficient function, e.g., atrophic muscle disorders. A better understanding of how nerve endings function may be attained by elucidation of the molecules and their interactions in these organelles.
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