6 research outputs found
Eukaryotic Assimilatory Nitrate Reductase Fractionates N and O Isotopes with a Ratio near Unity
In order to (i) establish the biological systematics
necessary
to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate
(<sup>15</sup>N/<sup>14</sup>N and <sup>18</sup>O/<sup>16</sup>O)
in the environment and (ii) investigate the potential for isotopes
to elucidate the mechanism of a key N cycle enzyme, we measured the
nitrate N and O isotope effects (<sup>15</sup>ε and <sup>18</sup>ε) for nitrate reduction by two assimilatory eukaryotic nitrate
reductase (eukNR) enzymes. The <sup>15</sup>ε for purified extracts
of NADPH eukNR from the fungus <i>Aspergillus niger</i> and
the <sup>15</sup>ε for NADH eukNR from cell homogenates of the
marine diatom <i>Thalassiosira weissflogii</i> were indistinguishable,
yielding a mean <sup>15</sup>ε for the enzyme of 26.6 ±
0.2‰. Both forms of eukNR imparted near equivalent fractionation
on N and O isotopes. The increase in <sup>18</sup>O/<sup>16</sup>O
versus the increase in <sup>15</sup>N/<sup>14</sup>N (relative to
their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and
1.09 ± 0.03 for NADH eukNR. These results are the first reliable
measurements of the coupled N and O isotope effects for any form of
eukNR. They support the prevailing view that intracellular reduction
by eukNR is the dominant step in isotope fractionation during nitrate
assimilation and that it drives the <sup>18</sup>ε:<sup>15</sup>ε ≈ 1 observed in phytoplankton cultures, suggesting
that this O-to-N isotope signature will apply broadly in the environment.
Our measured <sup>15</sup>ε and <sup>18</sup>ε may represent
the intrinsic isotope effects for eukNR-mediated N–O bond rupture,
a potential constraint on the nature of the enzyme’s transition
state
The contamination of commercial 15N2 gas with 15N-labeled nitrate/nitrite and ammonium and consequences for nitrogen fixation measurements
<p>The data contained herein are associated with the manuscript, "The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements", which is available at PLOS ONE.</p>
<p>We report on the presence of 15N-labeled nitrate/nitrite, ammonium, and nitrous oxide in commercial 15N2 gas and the enrichment of particulate nitrogen as a result of the biological assimilation of 15N labeled contaminants.</p
The δ<sup>15</sup>N of particulate nitrogen (δ<sup>15</sup>N<sub>PN</sub>) of <i>D. tertiolecta</i> harvested in stationary phase following growth in media containing sodium nitrate (and no ammonium) and equilibrated with <sup>15</sup>N<sub>2</sub> gas from Sigma Aldrich or Cambridge Isotopes.
<p>n = the number of experimental replicates.</p
(a) δ<sup>15</sup>N<sub>NH4</sub> (log scale) of 5 µmol L<sup>−1</sup> ammonium solutions after equilibration with 0.1 mL <sup>15</sup>N<sub>2</sub> gas from respective Sigma-Aldrich and Cambridge Isotopes lecture bottles <i>vs.</i> control solutions.
<p>Sigma-Aldrich treatments utilized 40 mL ammonium solutions, whereas Cambridge Isotopes treatments utilized 100 mL ammonium solutions. (<b>b</b>) δ<sup>15</sup>N<sub>NH4</sub> of higher sensitivity equilibrations of 5 µmol L<sup>−1</sup> ammonium solutions (10 mL) with 2.0 mL <sup>15</sup>N<sub>2</sub> gas from Cambridge Isotopes lecture bottles <i>vs.</i> control solutions. n = the number of experimental replicates.</p
(<b>a</b>) δ<sup>15</sup>N<sub>NO3+NO2</sub> (log scale) of nitrate solutions (10–300 µmol L<sup>−1</sup>) following equilibration with 0.1 mL <sup>15</sup>N<sub>2</sub> gas from lecture bottles procured from three distributors.
<p>Solutions were 40 mL for Sigma-Aldrich and Campro Scientific equilibrations, and 100 mL for Cambridge Isotopes equilibrations. The solid line corresponds to the δ<sup>15</sup>N<sub>NO3</sub> of the control solutions for Sigma-Aldrich and Cambridge Isotopes experiments (δ<sup>15</sup>N<sub>NO3</sub> = 23.5±0.5‰); the dashed line corresponds to controls for Campro Scientific experiments (δ<sup>15</sup>N<sub>NO3</sub> = 14.15±0.1‰). Paired symbols identify replicate experimental treatments. (<b>b</b>) Corresponding apparent δ<sup>18</sup>O<sub>NO3+NO2</sub> of the experimental nitrate solutions. The solid line corresponds to the δ<sup>18</sup>O<sub>NO3</sub> of control solutions for the Sigma-Aldrich and Cambridge Isotope experiments (δ<sup>18</sup>O<sub>NO3</sub> = 18.9±0.3‰); the dashed line corresponds to controls for Campro Scientific experiments (25.4±0.3‰).</p
(a) δ<sup>15</sup>N<sub>NO3+NO2</sub> (log scale) of higher sensitivity equilibrations of 10 µmol L<sup>−1</sup> nitrate solutions (10 mL) with 2 mL of <sup>15</sup>N<sub>2</sub> gas from a Cambridge Isotopes or a Sigma-Aldrich bottle.
<p>(<b>b</b>) Corresponding apparent δ<sup>18</sup>O<sub>NO3</sub> (log scale) of higher sensitivity equilibrations of the two stocks. n = the number of experimental replicates.</p