PMID- 26059529
OWN - NLM
STAT- MEDLINE
DCOM- 20160309
LR  - 20150805
IS  - 1097-0061 (Electronic)
IS  - 0749-503X (Linking)
VI  - 32
IP  - 8
DP  - 2015 Aug
TI  - Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under 
      anaerobic conditions, using alcohol dehydrogenase as sensor reaction.
PG  - 541-57
LID - 10.1002/yea.3078 [doi]
AB  - With the current quantitative metabolomics techniques, only whole-cell 
      concentrations of NAD and NADH can be quantified. These measurements cannot 
      provide information on the in vivo redox state of the cells, which is determined 
      by the ratio of the free forms only. In this work we quantified free NAD:NADH 
      ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and 
      the lumped reaction of glyceraldehyde-3-phosphate dehydrogenase and 
      3-phosphoglycerate kinase as sensor reactions. We showed that, with an 
      alternative accurate acetaldehyde determination method, based on rapid sampling, 
      instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and 
      quantification with HPLC, the ADH-catalysed oxidation of ethanol to acetaldehyde 
      can be applied as a relatively fast and simple sensor reaction to quantify the 
      free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of 
      ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in 
      anaerobic glucose-limited chemostats under steady-state and dynamic conditions. 
      The results found in this study showed that the cytosolic redox status (NAD:NADH 
      ratio) of yeast is at least one order of magnitude lower, and is thus much more 
      reduced, under anaerobic conditions compared to aerobic glucose-limited 
      steady-state conditions. The more reduced state of the cytosol under anaerobic 
      conditions has major implications for (central) metabolism. Accurate 
      determination of the free NAD:NADH ratio is therefore of importance for the 
      unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic 
      reversibility of each redox reaction.
CI  - Copyright (c) 2015 John Wiley & Sons, Ltd.
FAU - Bekers, K M
AU  - Bekers KM
AD  - Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
AD  - Department of Biotechnology, Delft University of Technology, Delft, The 
      Netherlands.
FAU - Heijnen, J J
AU  - Heijnen JJ
AD  - Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
AD  - Department of Biotechnology, Delft University of Technology, Delft, The 
      Netherlands.
FAU - van Gulik, W M
AU  - van Gulik WM
AD  - Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
AD  - Department of Biotechnology, Delft University of Technology, Delft, The 
      Netherlands.
LA  - eng
PT  - Journal Article
DEP - 20150714
PL  - England
TA  - Yeast
JT  - Yeast (Chichester, England)
JID - 8607637
RN  - 0 (Saccharomyces cerevisiae Proteins)
RN  - 0U46U6E8UK (NAD)
RN  - 3K9958V90M (Ethanol)
RN  - EC 1.1.1.1 (Alcohol Dehydrogenase)
RN  - IY9XDZ35W2 (Glucose)
SB  - IM
MH  - Alcohol Dehydrogenase/chemistry/*metabolism
MH  - Anaerobiosis
MH  - Ethanol/metabolism
MH  - Glucose/metabolism
MH  - NAD/analysis/*metabolism
MH  - Oxidation-Reduction
MH  - Saccharomyces cerevisiae/chemistry/enzymology/*metabolism
MH  - Saccharomyces cerevisiae Proteins/chemistry/*metabolism
OTO - NOTNLM
OT  - Saccharomyces cerevisiae
OT  - acetaldehyde
OT  - alcohol dehydrogenase
OT  - anaerobiosis
OT  - free NAD:NADH ratio
EDAT- 2015/06/11 06:00
MHDA- 2016/03/10 06:00
CRDT- 2015/06/11 06:00
PHST- 2014/09/01 00:00 [received]
PHST- 2015/05/11 00:00 [revised]
PHST- 2015/05/17 00:00 [accepted]
PHST- 2015/06/11 06:00 [entrez]
PHST- 2015/06/11 06:00 [pubmed]
PHST- 2016/03/10 06:00 [medline]
AID - 10.1002/yea.3078 [doi]
PST - ppublish
SO  - Yeast. 2015 Aug;32(8):541-57. doi: 10.1002/yea.3078. Epub 2015 Jul 14.