Radioactive labeling of the adenine nucleotide pool of cells as a method to distinguish among intracellular compartments. Studies on human platelets

Abstract

The study of adenine nucleotide metabolism is complicated by cellular nucleotide compartmentalization. In platelets, we have been able to use radioactive labeling with adenine to measure, precisely and accurately, changes in the cytoplasmic/mitochondrial pool of adenine nucleotides inspite of the fact that 60% of adenine nucleotides are present in amine-storing granules. High performance liquid chromatography was used to measure the concentrations of ATP, ADP, AMP and IMP from [14C]adenine-labeled platelets under conditions where the granule pool was absent. The radioactive measurements were directly proportional to the chemical measurements. Ethanol-insoluble, actin-bound ADP also had the same specific radioactivity as other metabolic adenine nucleotides. Since this pool can be directly separated from the bulk of cellular nucleotides, its specific radioactivity can be easily measured and used to calculate the concentration of each cytoplasmic adenine nucleotide from its measured radioactivity. These methods may be applicable to other cells.