Inositol trisphosphate (IP3) has recently been shown to stimulate Ca2+ release in a variety of cell types and therefore mediates such processes as muscle contraction, hormone secretion, and liver metabolism. In order to investigate the mechanism of IP3 action and to develop therapeutic drugs which act by duplicating or inhibiting IP3 action, one essential line of research is to study IP3 binding to its receptor. Such receptor binding studies require radioactively labeled IP3 with a high specific activity. The method proposed here would produced 32P3-IP3 with a specific activity greater than 10-fold higher than what is now available commercially or reported in scientific literature. The proposed protocol will use a readily available enzyme and 32P-ATP to label the precursor of IP3; therefore purified phosphatidylinositol phosphate will be labeled to produce 32P-phosphatidylinositol bisphosphate (32P-PIP2). 32P-PIP2 will then be hydrolyzed to produce 32P-IP3, which will be purified, and its specific activity will then be determined. The longer term (Phase II) objectives are to optimize the procedure and verify the utility of 32P-IP3 in receptor studies. 32P-IP3 is expected to be utilized both in basic research and in evaluating potential therapeutic drugs which act as IP3 agonists or antagonists.
