Assay of ATP in intact cells of the facultative phototroph Rhodobacter capsulatus expressing recombinant firefly luciferase.
This study reports on the construction, calibration and use of recombinant cells of Rhodobacter capsulatus expressing the luciferase gene of the North American firefly Photinus pyralis to detect, by bioluminescence, variations of endogenous ATP levels under various physiological conditions. We show that the antibiotic polymyxin B allows luciferin to rapidly move into cell cytosol, but does not make external ATP freely accessible to intracellular luciferase. Notably, in toluene:ethanol-permeabilized cells, the apparent K(mATP) for luciferase (50 microM) is similar to that measured in soluble cell fractions. This finding limits the applicability of the firefly luciferase for monitoring intracellular maximal ATP concentration because dark/aerobic-grown recombinant cells of Rba. capsulatus contain approximately 1.3-2.6+/-0.5 mM ATP. Therefore, the effects of chemical and physical factors such as oxygen, light, carbonyl cyanide m-chlorophenyl hydrazone and antimycin A on ATP synthesis were examined in cells subjected to different starvation periods to reduce the endogenous ATP pool below the luciferase ATP saturation level (< or =0.2 mM). We conclude that the amount of endogenous ATP generated by light is maximal in the presence of oxygen, which is required to optimize the membrane redox poise.
Di Tomaso
G
G
Department of Biology, University of Bologna, 42 Irnerio, 40126 Bologna, Italy.
Borghese
R
R
Zannoni
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D
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001419:Bacteria / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001517:Coleoptera / Q000201:enzymology*; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D011112:Polymyxin B / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases; D011112:Polymyxin B