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

Journal Article · Wed Feb 18 04:00:00 EST 2009 · Yeast, vol. 26, N/A, April 27, 2009, pp. 363-369

OSTI ID:966233

Sporty, J; Lin, S; Kato, M; Ognibene, T; Stewart, B; Turteltaub, K; Bench, G

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.

• Research Organization: Lawrence Livermore National Laboratory (LLNL), Livermore, CA
• Sponsoring Organization: USDOE
• DOE Contract Number: W-7405-ENG-48
• OSTI ID: 966233
• Report Number(s): LLNL-JRNL-410719
• Journal Information: Yeast, vol. 26, N/A, April 27, 2009, pp. 363-369, Journal Name: Yeast, vol. 26, N/A, April 27, 2009, pp. 363-369 Vol. 26
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BIOSYNTHESIS
CULTURE MEDIA
DETECTION
FUNCTIONALS
GLUCOSE
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
NAD
NICOTINIC ACID
SACCHAROMYCES CEREVISIAE
STRAINS
TRYPTOPHAN
YEASTS