The major, long term objective of this laboratory is to develop active site directed photoaffinity probes for important nucleotides and to expand this to include polynucleotides of known sequence which contain photoactive bases at specific locations. Photoaffinity probes are useful when they interact with enzymes either behaving as biological mimics or inhibitors of the effect produced by the natural compound. In this proposal, the following enzyme systems will be studied using previously synthesized photoprobes as well as new photoprobes not previously reported. The energetics of activation of Adenylate cyclase (AC) will be studied using Alpha32P and Gamma32P labeled 8N-3GTP (mimic of GTP) and various probes of TNP-GTP (a potent fluorescent inhibitor of AC) modified to contain a photoactive group and non-hydrolyzable phosphates. Also, [32P]8N3NAD will be used with cholera toxin and islet activating protein to attempt to 8N-3ADP-ribosylate the stimulatory and inhibitory GTP regulatory proteins of AC. Similar studies will be done on the ras oncogene product, p21. The catalytic subunit of type II cAMP dependent protein kinase (C-II) will be studied with regards to its autophosphorylation and photolabeling by [Gamma32P]8N-3dATP which appears to be more effective at autophosphorylation than [Gamma32P]ATP. Similar studies will be done on the cGMP dependent kinase, 3-PGA, pyruvate and creatine kinases. Newly synthesized 5-N3-dUTP is enzymatically incorporated by PolI into DNA in place of dTTP resulting in photoactive DNA. Similar 5-N-3U probes will be tested as substrates and active site probes of AMV reverse transcriptase and other DNA and RNA polymerases. 5N3-UDPG will be used to study glycogen synthetase. Both chemical and enzymatic synthesis of photoactive polynucleotides sequences will be done. The most obvious health related application of this research is the detection of viral induced proteins, some of which are oncogene protein products. Other uses are the detection of enzymes that are potential markers of neoplasia such as terminal deoxynucleotidyl transferase, adenosine deaminase, thymidlate kinase, etc., using Mug quantities of tissue or cells.
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