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'-AMP), an intracellular P-site inhibitor of adenylate cyclase, in rat liver mitochondria, 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 when using a reversed-phase HPLC column with a fluorescent-reaction, as established in this study. By using the optimized assay method developed here, 3'-AMP forming enzyme activity 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 no divalent cation was required for activity. From these results, 3'-AMP forming enzyme(s) in rat liver mitochondria could be classified as acid exoribonuclease, 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.