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Title: Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

Abstract

Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggestingmore » that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD{sup +} or NADH for lifespan extension under CR.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
966233
Report Number(s):
LLNL-JRNL-410719
TRN: US200921%%330
DOE Contract Number:  
W-7405-ENG-48
Resource Type:
Journal Article
Journal Name:
Yeast, vol. 26, N/A, April 27, 2009, pp. 363-369
Additional Journal Information:
Journal Volume: 26
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BIOSYNTHESIS; CULTURE MEDIA; DETECTION; FUNCTIONALS; GLUCOSE; HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY; NAD; NICOTINIC ACID; SACCHAROMYCES CEREVISIAE; STRAINS; TRYPTOPHAN; YEASTS

Citation Formats

Sporty, J, Lin, S, Kato, M, Ognibene, T, Stewart, B, Turteltaub, K, and Bench, G. Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae. United States: N. p., 2009. Web.
Sporty, J, Lin, S, Kato, M, Ognibene, T, Stewart, B, Turteltaub, K, & Bench, G. Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae. United States.
Sporty, J, Lin, S, Kato, M, Ognibene, T, Stewart, B, Turteltaub, K, and Bench, G. Wed . "Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae". United States. https://www.osti.gov/servlets/purl/966233.
@article{osti_966233,
title = {Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae},
author = {Sporty, J and Lin, S and Kato, M and Ognibene, T and Stewart, B and Turteltaub, K and Bench, G},
abstractNote = {Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD{sup +} or NADH for lifespan extension under CR.},
doi = {},
url = {https://www.osti.gov/biblio/966233}, journal = {Yeast, vol. 26, N/A, April 27, 2009, pp. 363-369},
number = ,
volume = 26,
place = {United States},
year = {2009},
month = {2}
}