Supplementary Fig. S1. Relative peak intensities of peak A (panel A), peak B (panel
B) and peak C (panel C) obtained when EA.hy926 human endothelial cells were
incubated with 0 – 500 µM of 15N4-ARG for 2 hours. Relative peak intensities of peak
A, B, and C were determined as peak area ratio against two different internal
standards, i.e., D4-CIT for peak A and 13C6-ARG for peak B and peak C. Data are
A
Peak A, peak area ratio
presented as mean ± SD (n=6).
0.8
0.6
0.4
0.2
0.0
0
100 200 300 400 500 600
B
Peak B, peak area ratio
Extracellular 15N4-ARG, M
1.0
0.8
0.6
0.4
0.2
0.0
0
100 200 300 400 500 600
C
Peak C, peak area ratio
Extracellular 15N4-ARG, M
0.020
0.015
0.010
0.005
0.000
0
100 200 300 400 500 600
Extracellular 15N4-ARG, M
Supplementary Fig. S2. Correlations between relative peak intensity of
15N-nitrite
(panel A) and relative peak intensity of
15N -ARG
3
15N -CIT
3
vs.
vs. 15N-nitrite (panel B)
after EA.hy926 human endothelial cells were incubated with 0 – 500 µM of 15N4-ARG
for 2 hours (n=6 for each concentration). The peak intensity of 15N3-ARG was
corrected for its presence as an impurity of
15N -ARG.
4
0.7
15
N3-CIT, peak area ratio
A
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
10
20
15
40
30
40
N-nitrite, nM
N3-ARG, peak area ratio
0.20
0.15
0.10
0.05
15
B
30
0.00
0
10
20
15
N-nitrite, nM
Supplementary Fig. S3. Correlations between 15N3-CIT vs. 15N-nitrite (black symbols;
p=0.862, R2=0.740) and 15N3-CIT+15N3-ARG vs. 15N-nitrite (red symbols; p=0.865,
R2=0,744) after EA.hy926 human endothelial cells were incubated with 0 – 500 µM
of 15N4-ARG for 2 hours (n=6 for each concentration, each point represents an
individual run). The solid lines represent the linear regression of each correlation
when the y-intercept was set at 0.
Estimated concentration, nM
700
600
15
N3-CIT
15
N3-CIT+15N3-ARG
500
400
300
200
100
0
0
10
20
15
N-Nitrite, nM
30
40
Supplementary Fig. S4. Production of 15N3-CIT and nitrite and nitrate in the cell
lysates when cell lysates were exposed to 0 – 100 µM of 15N4-ARG for 30 min (n=3
for each concentration). 15N3-CIT were measured as described in the materials and
methods and nitrite and nitrate concentrations were determined by Ultrasensitive
Colorimetric Assay for Nitric Oxide Synthase kit (Oxford Biomedical Research, Inc.,
v, pmol/mg/min
Oxford, MI).
120
100
80
60
40
20
15
N3-CIT
Nitrite+Nitrate
4
2
0
0
20
40
60
80
15N -ARG concentration, M
4
100
120
Supplementary Fig. S5. Schematic representation of metabolic fates of
14C-ARG.
The radioisotope labeling is set on the ureido-carbon atom. It is noted that
CIT→ARG recycling does not generate any additional 14C-CIT, although 14C-ARG is
re-generated along with unlabeled NO. ASS; Argininosuccinate synthase, ASL;
argininosuccinate lyase.
14C-argininosuccinate
Aspartate
14C-ARG
14C-CIT
Fumarate
14C-ARG
Supplementary Fig. S6. Stability of 15N4-ARG (A) in the cell incubation medium and
(B) cell lysate mixture. 15N4-ARG concentrations were determined at 0 – 120 min
incubation in the blank cell incubation medium or cell lysate.
Supplementary Fig. S7. Production of 15N3-CIT (A) in the cell lysates when cell
lysates were incubated with 10 µM of 15N4-ARG and (B) in the intact cells when cells
were incubated with 100 µM 15N4-ARG.
A
4
3
2
1
15
15 3
N3-CIT, pmol/mg protein
N -CIT, pmol/mg protein
5
0
0
10
20
30
40
50
60
Time,min
min
Time,
120
15
15
N
protein
N33-CIT,
-CIT,pmol/mg
pmol/mgprotein
B
100
80
60
40
20
0
0
20
40
60
80
min
Time, min
100
120
70