Determination of intracellular pyridine nucleotide levels by bioluminescence using anaerobic bacteria as a model.
An assay for the determination of NAD(P)+ and NAD(P)H in extracts from the obligate anaerobe bacterium Thermoanaerobacter finnii is developed and the strategy for this development is described. This assay performed with constant FMN reductase (EC 1.6.8.1) and luciferase (EC 1.14.14.3) concentrations has been shown to detect as low as 1 pmol pyridine nucleotide. With this assay recovery, efficiency of extraction, reliability, and detection limit of the procedures were determined. To our knowledge this is the first bioluminometric assay for the determination of pyridine nucleotide levels in bacterial extracts.
Schmid
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Institut für Biotechnologie 1 der KFA Jülich GmbH, Federal Republic of Germany.
Schimz
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KL
Sahm
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H
D000227:Adenine Nucleotides / Q000032:analysis; D001421:Bacteria, Anaerobic / Q000378:metabolism*; D038181:FMN Reductase; D005285:Fermentation; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D008954:Models, Biological; D009243:NAD / Q000032:analysis*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP / Q000032:analysis*; D014994:Xylose / Q000378:metabolism
D000227:Adenine Nucleotides; D009243:NAD; D009249:NADP; D014994:Xylose; D008156:Luciferases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases