The general objective of this proposal is to study protein-nucleotide interactions in cell preparations, membranes, organelles and purified enzyme systems using nucleotide photoaffinity analogs. The successful synthesis and utilization of several photoaffinity analogs (e.g. 8-N3cAMP, 8-N3GTP, etc.) have already been accomplished in our laboratory and are being productively used by ourselves and other researchers. Several of these analogs are now commercially available and others will follow soon. Herein, we first propose to perform research directed toward better utilization of these probes with biological systems in various stages of purification. While we know that these probes cannot resolve all questions concerning nucleotide regulation of biological phenomenon they can give specific insights otherwise unavailable or very difficult to obtain. For example, these probes may be used to determine: (1) active site time dependent chemical events such as phosphate hydrolysis in situ (e.g. on active site N3GTP hydrolysis during tubulin polymerization), (2) cellular location of specific nucleotide binding proteins, (3) time of appearance of a specific nucleotide receptor protein in a cellular system (caused by developmental processes; stage of cell life cycle, viral infection, etc.), (4) unoccupied cAMP site analysis, and (5) which vectors (allosteric interactions, phosphorylation, antophosphorylation, competitive binding, etc.) affect specific nucleotide binding to receptor proteins and thereby modulate nucleotide regulation of certain biological phenomenon. Secondly, we propose the synthesis of new nucleotide photoaffinity probes. We are currently synthesizing photoprobes for guanosine """"""""magic spot"""""""" compounds (e.g. pp 8-N3Gpp) and like adenosine compounds. Also, we propose the synthesis of two new classes of nucleotide probes called """"""""multisubstrate photoaffinity probes"""""""" and """"""""transition state photoaffinity probes"""""""". These classes of probes combine the structural features of two substates in a single molecule which also contains a photoreactive group. Respective examples would be 8-N3GDP-mannose and the 8-azidozdenosine derivatives of P1, P5-diadenosine pentaphosphate (Ap5A) a very strong competitive inhibitor of adenylate kinases.
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