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NAD+ and NADH Kinetics, using 13C NMR

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SEEK ID: https://demo.fairdomhub.org/assays/9

Experimental Assay

Katy Wolstencroft

Projects: SysMO DB

Investigation: Glycolitic Flux in Lactococcus lactis

Study: The effects of Redox Charge on Glycolytic Flux

Assay type: Glucose Pulse

Technology type: NMR

Organisms: Escherichia coli

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Created: 8th Aug 2011 at 15:52

Last updated: 26th Oct 2017 at 11:57

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Katy Wolstencroft adminasset_housekeeper

Projects: SysMO DB, SulfoSys

Institutions: School of Computer Science, University of Manchester, University of Amsterdam

SysMO DB

The main objectives of SysMO-DB are to: facilitate the web-based exchange of data between research groups within- and inter- consortia, and to provide an integrated platform for the dissemination of the results of the SysMO projects to the scientific community. We aim to devise a progressive and scalable solution to the data management needs of the SysMO initiative, that:

* facilitates and maximises the potential for data exchange between SysMO research groups;
* maximises the ‘shelf life’ and
...

Programme: SysMO

Public web page: http://www.sysmo-db.org/

Glycolitic Flux in Lactococcus lactis
SysMO DB

An investigation into the control mechanisms of glycolytic flux in Lactococcus lactis.

This investigation represents the data and models published by Hoefnagel et al in JWS Online and in M.H.N. Hoefnagel, J. Hugenholtz and J.L. SnoepMol. Biol. Reports 29, 157-161 (2002)

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The effects of Redox Charge on Glycolytic Flux
SysMO DB

A study which combines mathematical modelling and experimental measurements to determine the effects of redox charge on glycolytic flux

Person responsible: Jacky Snoep

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Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR
SysMO DB

Abstract (Expand)

The involvement of nicotinamide adenine nucleotides (NAD(+), NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using (13)C and (31)P NMR to monitor in vivo the kinetics … of the pools of NAD(+), NADH, ATP, inorganic phosphate (P(i)), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of (13)C-labeled NAD(+) and NADH. The capacity of L. lactis MG1363 to regenerate NAD(+) was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH(-) deficient strain was constructed by double crossover. Upon supply of glucose, NAD(+) was constant and maximal (approximately 5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH(-) strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH(-) strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD(+). However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P(i) content could play an important role.

Authors: Ana Rute Neves, Rita Ventura, Nahla Mansour, Claire Shearman, Michael J Gasson, Christopher Maycock, Ana Ramos, Helena Santos

Date Published: 13th May 2002

Journal: J. Biol. Chem.

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