NAMPT and NAPRT activities in the absence or presence of ATP.

<p>NAMPT (15 ng) and NAPRT (20 ng) were incubated with 20 µM [¹⁴C]Nam and [¹⁴C]NA for 30 min, respectively, in the presence of 1 µM PRPP (low substrate concentration) without or with 1 mM ATP. NAMPT (96 ng) and NAPRT (100 ng) were also incubated with 50 µM [¹⁴C]Nam and [¹⁴C]NA for 7 and 6 min, respectively, in the presence of 50 µM PRPP (high substrate concentration) without or with 1 mM ATP.</p

ATP is an essential activator of NAMPT and NAPRT reactions.

<p>NAMPT (A, 15 ng) and NAPRT (B, 20 ng) were incubated with 20 µM [¹⁴C]Nam and [¹⁴C]NA for 6 and 7 min, respectively, in the presence of 1 µM PRPP and indicated concentrations of ATP. The amounts of NMN (A) and NaMN (B) formed were determined. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022781#pone-0022781-g002" target="_blank">figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022781#pone-0022781-g003" target="_blank">3</a> are representative of at least three experiments.</p

Metabolic pathways of the salvage NAD synthesis.

<p>NaAD, NA adenine dinucleotide; <i>NMNAT</i>, NMN adenylyltransferase; <i>NaMNAT</i>, NaMN adenylyltransferase; <i>NADsyn</i>, NAD synthetase. Broken arrows indicate the possible extracellular pathway in NAD biosynthesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022781#pone.0022781-Imai1" target="_blank">[14]</a>.</p

Kinetic parameters in the NAMPT and NAPRT reactions.

<p>NAMPT (36 ng) was incubated with various Nam concentrations at a fixed PRPP concentration (300 µM) for 7 and 20 min in the presence or absence of 1 mM ATP, respectively. The enzyme (7 and 36 ng) was also incubated with various concentrations of PRPP at fixed Nam concentrations (5 and 40 µM) for 7 and 30 min in the presence or absence of 1 mM ATP, respectively. NAPRT (70 and 100 ng) was incubated with various NA concentrations at a fixed PRPP concentration (300 µM) in the presence or absence of 1 mM ATP, respectively, for 30 min. The enzyme (12 and 90 ng) was also incubated with various concentrations of PRPP at fixed NA concentrations (50 and 380 µM) for 15 and 30 min in the presence or absence of 1 mM ATP, respectively. The mutant NAPRT (H213N-NAPRT, 140 ng) was incubated with various NA concentrations at a fixed PRPP concentration (600 µM) or with various concentrations of PRPP at a fixed NA concentration (650 µM) in the presence or absence of 1 mM ATP for 60 min. <i>K_m</i> and <i>V_max</i> values represent the mean ± S.D. of at least three separate experiments.</p

PRPP, ATP, and NMN are degraded in mouse blood plasma.

<p>PRPP, ATP, or NMN (150, 300, or 60 pmol, respectively) was incubated with the blood plasma (30 µl) in the presence of EDTA/EGTA at 4 (<i>circles</i>) or 37°C (<i>triangles</i>) or in the absence of the chelators at 37°C (<i>squares</i>) for the indicated times. After the incubation, the amounts of PRPP (A), ATP (B), and NMN (C) remaining at each time point in the plasma were determined and expressed as percent of those at 0 min.</p