A specific and rapid method for determination of adenosine 3 '-monophosphate (3 '-AMP) content and 3 '-AMP forming enzyme activity in rat liver mitochondria, using reversed-phase HPLC with fluorescence detection

To study the physiological significance of adenosine 3'-monophosphate (3'-A MP), an intracellular P-site inhibitor of adenylate cyclase, in rat liver m itochondria, a specific, rapid and reliable assay method for determination of 3'-AMP and the activity of its forming enzyme is required. 3'-AMP in rat liver was determined to be ca, 23+/-7 nmol/g wet weight, but no 2-deoxy-3' -AMP, another P-site inhibitor of adenylate cyclase, was detected, even whe n using a reversed-phase HPLC column with a fluorescent-reaction, as establ ished in this study. By using the optimized assay method developed here, 3'-AMP forming enzyme a ctivity in rat crude mitochondrial extract was found to be enhanced by EDTA and inhibited by p-chloromercurybenzoate. The optimum pH was ca, 5.8 and n o divalent cation was required for activity, From these results, 3'-AMP for ming enzyme(s) in rat liver mitochondria could be classified as acid exorib onuclease, which mainly existed in an active form. The results obtained in this study will help to gain more insight into the physiological roles of 3 '-AMP in living systems.