The reagents and reaction mixture were combined according to the protocol by Molecular Probes however, the volume of each assay was increased to 1,000 μl to account for the ∼100-fold stoichiometric increase in the quantum yield due to the higher optical density (OD) readings for the production of ATP relative to the content. The ATP determination kit (A-22066) by Molecular Probes (Eugene, OR) containes d-luciferin, luciferase, dithiothreitol (DTT), ATP, and a reaction buffer (10 ml of 500 mM tricine buffer, pH 7.8, 100 mM MgSO 4, 2 mM EDTA, and 2 mM sodium azide). Upon addition, ATP combines with luciferin to form luciferyl adenylate and inorganic pyrophosphate (PP i) on the surface of the luciferase enzyme as shown in reaction 1Ĭhemicals. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. To study ATP content and production from isolated mitochondria, we used a commercially available ATP determination kit.
#ATP PRODUCTION FREE#
The large free energy released during the hydrolysis of ATP is available for immediate use (i.e., muscle contraction, chemiosmotic homeostasis, and normal cellular function) or can be stored for future cellular activity, such as protein synthesis, genomic replication, and cell growth ( 37, 43). Because ATP is tightly bound to the F 1 head in the mitochondrial membrane, a large amount of free energy is released during the rapid hydrolysis of ATP to ADP or AMP. The catalytic sites on the F 1 head of the ATP synthase complex, which projects onto the protochemically negative matrix side (N-phase) of the membrane, contain three nucleotide binding sites that undergo a multiphase, catalytic cycle to produce ATP ( 43) from ADP and phosphate (i.e., oxidative phosphorylation). Cellular function is maintained through the hydrolysis of ATP to ADP.
#ATP PRODUCTION SERIES#
The inner membrane of the mitochondria contains a complex series of enzymes and other proteins that are involved in the catabolism of food molecules to liberate metabolically useful energy in the form of ATP. Mitochondria produce ∼90% of the required energy necessary for cellular function ( 46). This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. For a 25 μM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. The luminescent signals of the reaction mixture and a 0.5 μM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function.