Abstract
As the principal co-factors of many metabolic pathways, the measurement of both adenine nucleotides and nicotinamide adenine dinucleotide provides important information about cellular energy metabolism. However, given their rapid and reversible conversion as well as their relatively low concentration ranges, it is difficult to measure these compounds. Here, we describe a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides in plants. In addition, nicotinamide adenine dinucleotide is a crucially important redox-active substrate for multiple catabolic and anabolic reactions with the ratios of NAD+ /NADH and NADP+ /NADPH being suggested as indicators of the general intracellular redox potential and hence metabolic state. Here, we describe highly sensitive enzyme cycling-based colorimetric assays (with a detection limit in the pmol range) performed subsequent to a simple extraction procedure involving acid or base extraction to allow the measurement of the cellular levels of these metabolites. © 2020 The Authors. Basic Protocol 1: Preparation of plant material for the measurement Basic Protocol 2: Measurement of ATP, ADP, and AMP via HPLC Basic Protocol 3: NAD+ /NADP+ measurements Basic Protocol 4: NADH/NADPH measurements Basic Protocol 5: Data analysis and quality control approaches.
Highlights
Both adenine nucleotides and nicotinamide adenine dinucleotide are essential for energy transfer as wells as enzyme regulation, functioning as co-factors in various signal transduction pathways in living plant cells (Atkinson, 1971; Srivastava & Bernhard, 1985)
Understanding Results The adenine nucleotide and nicotinamide adenine dinucleotide measurement protocols presented in this article have been used by us and our collaborators to characterize the levels of these metabolites
The nicotinamide adenine dinucleotide (NAD)+/NADH ratio was found to be changed in mutant lines while the Nicotinamide adenine dinucleotide phosphate (NADP)+/NADPH ratio was not found to change (Zhang et al, 2018)
Summary
To cite this version: Youjun Zhang, Ina Krahnert, Antje Bolze, Yves Gibon, Alisdair Fernie. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Basic Protocol 1: Preparation of plant material for the measurement Basic Protocol 2: Measurement of ATP, ADP, and AMP via HPLC Basic Protocol 3: NAD+/NADP+ measurements Basic Protocol 4: NADH/NADPH measurements Basic Protocol 5: Data analysis and quality control approaches. How to cite this article: Zhang, Y., Krahnert, I., Bolze, A., Gibon, Y., & Fernie, A. Current Protocols in Plant Biology, 5, e20115.
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