PROCEDURE TO PRODUCE HIGH-SPECIFIC-ACTIVITY (32)P-IP3

INOSITOL TRISPHOSPHATE (IP3) HAS RECENTLY BEEN SHOWN TO STIMULATE CA(2+) 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 THAT 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 PRODUCE (32)P-IP3 WITH A SPECIFIC ACTIVITY GREATER THAN 10-FOLD HIGHER THAN WHAT IS NOW AVAILABLE COMMERCIALLY OR REPORTED IN SCIENTIFICLITERATURE. THE PROPOSED PROTOCOL WILL USE A READILY AVAILABLE ENZYME AND (32)P-ATP TO LABEL THE PRECURSOR OF IP3; THEREFORE, PURIFIED PHOSPHATIDYLINOSITOL PHOSPHATE WILLBE LABELED TO PRODUCE (32)P-PHOSPHATIDYLINOSITOL BISPHOSPHATE ((32)P-PIP2). (32)P-PIP2 WILL THEN BE HYDROLYZED TO PRODUCE (32)P-IP3, WHICH WILL BE PURIFIED. ITS SPECIFIC ACTIVITY WILL THEN BE DETERMINED. THE LONGER-TERM (PHASE II) OBJECTIVES ARE TO OPTIMIZE THE PROCEDURE AND TO VERIFTY THE UTILITY OF (32)P-IP3 IN RECEPTOR STUDIES. (32)P-IP3 IS EXPECTED TO BE UTILIZED BOTH IN BASIC RESEARCH AND IN EVALUATING POTENTIAL THERAPEUTIC DRUGS THAT ACT AS IP3 AGONISTS OR ANTAGONISTS.