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Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
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Org. Biomol. Chem.
, 2012, 10, 323
www.rsc.org/obc PAPER
Tyrosine nitration affects thymidylate synthase properties
El˙
zbieta Da˛browska-Ma´
s,†aTomasz Fra˛czyk,†aTomasz Ruman,bKarolina Radziszewska,cPiotr Wilk,a
Joanna Cie´
sla,aZbigniew Zieli´
nski,aAgata Jurkiewicz,dBarbara Gołos,aPatrycja Wi´
nska,a
El˙
zbieta Wałajtys-Rode,eAndrzej Le´
s,dJoanna Nizioł,bAdam Jarmuła,aPiotr Stefanowicz,c
Zbigniew Szewczukcand Wojciech Rode*a,b
Received 10th August 2011, Accepted 20th September 2011
DOI: 10.1039/c1ob06360j
Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental
and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with
anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and
tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated
in vitro inthepresenceofNaHCO
3,NaNO
2and H2O2at pH 7.5, underwent tyrosine nitration, leading
to a Vmaxapp 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS)
tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not
distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula
H2N-Gly-X-Gly-COOH (X =Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR
spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH
peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement
with the strongest peak found in 15N-1HHMBCspectrumofnitratedprotein.MSanalysisofnitrated
human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing
nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized
cysteine (human protein Cys210, with catalytically critical Cys195 remaining apparently unmodified)
residues.
Introduction
Thymidylate synthase (TS; EC 2.1.1.45), a target in chemotherapy
of a number of diseases, including cancer,1catalyzes the N5,10-
methylenetetrahydrofolate (meTHF)-assisted C(5)-methylation of
dUMP,2required for DNA synthesis. It is, consequently, of
interest to examine possible post-translational modifications of
the enzyme in living cells.
Nitration of protein tyrosine residues is a post-translational
modification, potentially affecting the function of a protein. It is
associated with more than 50 diseases, including cancer, involving
intensified NO biosynthesis.3The modification in vivo appears
to be selective, with not many proteins becoming nitrated and
aNencki Institute of Experimental Biology, Polish Academy of Sciences,
Warszawa, Poland. E-mail: rode@nencki.gov.pl; Fax: (+48-22) 822 5342
bRzesz´
ow University of Technology, Faculty of Chemistry, Rzesz´
ow, Poland
cFaculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie Street,
50-383, Wrocław, Poland
dFaculty of Chemistry, University of Warsaw, 1 L. Pasteur Street, 02-093,
Warszawa, Poland
eUniversity of Information Technology and Management in Rzesz´
ow, Chair
of Cosmetology, 2 Sucharskiego Street, 35-225, Rzesz´
ow, Poland
† These authors contributed equally to this work.
only very few residues being modified in each protein. Moreover,
even with good nitration targets, the yield of protein nitro-tyrosine
formation is low.4Nevertheless, the few known examples show that
nitration of one or two tyrosine residues is enough to cause loss
or gain of function (for physicochemical consequences of protein
tyrosine nitration, cf . ref. 4), suggesting a need for studies directed
at protein structure-function analysis of specific proteins found to
undergo nitration in vivo.5
As tetranitromethane nitration of sulfhydryl-blocked Lac-
tobacillus casei thymidylate synthase protein caused enzyme
inactivation,6it was of interest to test the possibility of TS tyrosine
nitration in animal cells/tissue, and to determine to what extent
enzyme properties might be affected by chemical nitration of
TS tyrosine. Therefore kinetic and physicochemical (NMR, MS)
studies were undertaken of in vitro nitrated preparations of human,
mouse and Caenorhabditis elegans recombinant TSs. In order
to enable interpretation of NMR resonances found in nitrated
protein spectra, model tripeptides of a general formula H2N-Gly-
X-Gly-COOH (X =Ph e, Tyr, Trp , Ly s, Ar g, Hi s, Se r, T hr, Cys, Gl y )
were nitrated and analyzed using NMR spectroscopy. Besides, the
theoretical calculations of 15N NMR chemical shift for models of
the nitrated tripeptides were performed and compared with those
found experimentally.
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Results and discussion
Reactivity of purified endogenous TS proteins to anti-tyrosine
antibodies
Highly purified TS proteins, isolated from calf thymus, and L1210
parental and FdUrd-resistant cells, were found to be nitrated (Fig.
1), based on a specific reaction with anti-nitrotyrosine antibodies
(Sigma-Aldrich, Anti-Nitro-tyrosine, Cat. No. N0409), suggesting
the enzyme to undergo this modification endogenously in normal
and tumor tissues. The reaction was specific to tyrosine, as the
presence of nitro-tyrosine (10 mM) in the buffer containing
anti-tyrosine antibodies (Fig. 1B), as well as the reduction
of nitro-tyrosine to amino-tyrosine with Na2S2O4(Fig. 1C),
prevented anti-tyrosine antibodies binding with the proteins. Of
note is that the lack of the signal in panels B and C (Fig. 1) was
not caused by an insufficient amount of protein, as demonstrated
by the incubation of the same PVDF membrane with anti-TS
antibodies (Fig. 1, E–F). It should be worthwhile to add
that, according to the manufacturer, the polyclonal antibodies
preparation used is fairly specific for protein nitro-tyrosine.
While it recognizes nitrated proteins and 3-nitro-L-tyrosine, it
does not cross-react with L-tyrosine, p-nitro-L-phenylalanine,
3-amino-L-tyrosine, 3-chloro-L-tyrosine and phospho-L-tyrosine
BSA conjugates (http://www.sigmaaldrich.com/life-science/
cell-biology/antibodies/learning-center/antibody-explorer/
spotlights/anti-nitrotyrosine.html).
A comparison of nitration levels observed for the three endoge-
nous enzyme preparations (Fig. 1, lanes 5–7) and that of the in
vitro nitrated mouse recombinant enzyme determined to contain
0.8 and 1.6 mol/mol of TS monomer (Fig. 1, lanes 2 and 3,
respectively), based on the ratio of signal intensities resulting from
application of anti-nitro-tyrosine and anti-thymidylate synthase
antibodies, indicates the modification level of the endogenous
proteins to be much lower and concern presumably only a small
fraction of each of those proteins.
Recombinant TS in vitro nitration and its effect on enzyme
properties
Each human, mouse and Ceanorhabditis elegans recombinant
TS preparation, incubated in vitro at pH 7.5 in the presence of
peroxynitrite (ONOO-) producing mixture, containing NaHCO3,
NaNO2and H2O2(1.00 : 1.05 : 1.00), underwent [H2O2]-dependent
tyrosine nitration (Fig. 1, lanes 2–4, and Fig. 2; Note:H
2O2concen-
tration reflects concentration of the peroxynitrite producing mix-
ture, its constituents present always in constant proportion). The
reaction dependence on H2O2concentrationvariedindifferentTS
proteins, being similar with the human (Fig. 2) and C. elegans
proteins (linear progress observed in the range of 10–70 mM
H2O2), but distinctly different with the mouse protein (linear
progress observed in the range of only 5–20 mM H2O2,withlower
nitration at higher concentrations). While this modification did not
distinctly influence the Kmvalues reflecting enzyme interactions
with dUMP and meTHF or the inhibition and inactivation rate
constants (not shown) reflecting slow-binding of TS by 5-fluoro-
dUMP (cf. ref. 7), it affected TS activity, leading to a Vmax app 2-
fold lower following nitration of 1 (with human or C. elegans
TS) or 2 (with mouse TS) tyrosine residues per monomer (not
shown). It should be mentioned that initial experiments, involving
Fig. 1 Nitro-tyrosine detection by specific antibodies in chemically ni-
trated mammalian recombinant TS and endogenous enzyme preparations
purified from tumour and normal tissues and separated by SDS-PAGE
(without 2-mercaptoethanol). Proteins were stained with SyproR
Rub y
Protein Gel Stain (G) or, following transfer to PVDF membrane,
underwent first reaction with anti-nitroY antibodies (A–C), followed by
removing of anti-nitroY antibodies and treatment with anti-TS antibodies
(D–F). Negative controls included treatment with anti-nitroY antibodies
either in the presence of 10 mM nitro-tyrosine (B) or following reduction
of nitro-tyrosine to amino-tyrosine with 100 mM Na2S2O4at pH 9,0 (C).
Nitrated BSA (positive control; lane 1), mouse recombinant TS nitrated
with 8 mM (0.8 mol nitroY/mol TS subunit; lane 2), 12 mM (1.6 mol
nitroY/mol TS subunit; lane 3) or 12 mM inactivated (negative control,
0 mol nitroY/mol TS; lane 4) peroxynitrite, and endogenous TS purified
from calf thymus (lane 5), and L1210 parental (lane 6) and FdUrd-resistant
(lane 7) cells. TS nitration presented in the bar chart was calculated as the
ratio of signals deriving from bands of nitrated protein (A) and TS protein
(D), with lighter and darker bars corresponding TS bands (marked with
lighter and darker arrows in A and B) showing lower and higher mobility,
respectively.
TS nitration with synthesized authentic peroxynitric acid, showed
the enzyme to undergo an instant inactivation (not shown), pre-
sumably due to the catalytic cysteine2modification. In accord, ap-
plication of the peroxynitrite producing mixture, including CO32-,
known to inhibit sulfhydryl oxidation and enhance nitration of
aromatics,8allowed to study nitration with the enzyme activity
preserved.
NMR analyses of nitrated model compounds
In order to enable interpretation of resonances found in nitrated
protein spectra, model compounds, including free amino acids
(Phe, Tyr and Trp) and tripeptides of a general formula H2N-Gly-
X-Gly-COOH (X =Ph e, Tyr, Trp , Ly s, Ar g, Hi s, Se r, T hr, Cys,
Gly), were nitrated and analyzed using NMR spectroscopy. While
the three free amino acids did not allow quantitative analysis
due to relatively low solubility of aromatic amino acids, such
a study was possible with the use of the tripeptides, serving as
simple models of proteins. The 15N chemical shifts resulting from
NMR studies, as well as DFT calculations performed for nitrated
forms of truncated amino acid moieties (Fig. 3), are presented
in Table 1. As the experimental NMR data clearly showed
formation of nitro-species only for H-Gly-Tyr-Gly-OH and
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Fig. 2 Chemical nitration of human (squares), mouse (circles) and C.
elegans (crosses) recombinant TS proteins: dependence of nitrated enzyme
catalytic potency, reflected by the Vmax app value, on H2O2concentration
in the reaction mixture (Note: H2O2concentration reflects concentration
of the peroxynitrite producing mixture, its constituents present always in
constant proportion). Vmaxapp value was determined at varying [dUMP]
and constant [N5,10-methylenetetrahydrofolate].
Fig. 3 Models of nitro-aminoacids used for DFT calculations: 1a
o-NO2Phe, 1b m-NO2Phe, 1c p-NO2Phe, 2a –2-NO
2Trp , 2b –4-NO
2Trp ,
2c –5-NO
2Trp , 2d –6-NO
2Trp , 2e –7-NO
2Trp , 3–3-NO
2Tyr, 4a
2-NO2-His N1-H, 4b –5-NO
2-His N1-H, 4c –2-NO
2-His N3-H, 4d
5-NO2-His N3-H, 5S-NO2Cys, 6O-NO2Ser, 7O-NO2Thr, 8a
N-w-NO2Arg, 8b N-e-NO2Arg, 9N-NO2Lys.
H-Gly-Phe-Gly-OH peptides, NMR-DFT comparison of
chemical shifts was possible for these two systems. However,
calculated 15N chemical shifts, with chemical shift differences
calculated as Dd=dexp15 N–dcal15 N(dexp15 N – experimental
chemical shift; dcal15 N – calculated chemical shift) for models of
nitrated H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides
(Table 1, column 5) taken into consideration, could be useful
for prediction of 15N chemical shifts of similar nitro-systems
or even nitro-proteins. Of note is that recently Lehnik and
Kirsch analyzed peroxynitrite nitration of L-tyrosine and related
compounds with the use of 15N CIDNP method.9
Nitration of the tyrosine-containing tripeptide resulted in the
formation of 3-nitro-tyrosine moiety, along with only traces of
other compounds (Fig. 4). Based on the 1H NMR data, the molar
ratio of tripeptide containing nitro-tyrosine to that containing
tyrosine was 1: 4.4. The confirmation of H-Gly-Tyr(15NO2)-Gly-
OH peptide formation was found in the 15N-1H HMBC spectrum
(Fig. 5), clearly showing the heteronuclear coupling between 15N-
nitrate group (373.4 ppm) and the adjacent aromatic hydrogen
atom at the ring C(2) (7.84 ppm). As indicated in Fig. 5, the 15N
resonance of the above mentioned nitro-moiety is a doublet, due
to the 3-bond proximity of the hydrogen atom at the ring C(2).
Of note are interesting observations resulting from studies
on the nitrated phenylalanine-containing tripeptide. The peaks
observed in the 15N-1H HMBC spectrum (not shown) suggest the
existence of two compounds, each containing nitro group attached
to the aromatic ring. The NMR data, describing the cross peak,
showing heteronuclear coupling between 15NO2nitrogen (371.2
ppm) and aromatic hydrogen atom (8.09 ppm), are in a very good
agreement with those for 4-nitrotoluene that contains identical 4-
nitrobenzyl moiety.10 Thus the latter pair of chemical shifts points
to the formation of H-Gly-Phe(NO2)-Gly-OH peptide, with the
15NO2group generally connected to electronically and sterically
favored para position.
The second cross peak (Fig. 5) foundin 15 N-1H HMBC spectrum
(7.88 ppm at H-axis and 374.1 ppm) is in a surprisingly good
agreement with our data found for H-Gly-Tyr(NO2)-Gly-OH (vide
supra). The NMR data strongly suggest the existence of nitro-
tyrosine moiety that must have been formed by nitration of tyro-
sine moiety. Consequently, the tyrosine moiety must have resulted
from phenylalanine moiety hydroxylation, a process described in
the literature.11,12 However, the yield of the overall process is very
low, as the molar ratio calculated from 1H NMR spectrum shows
the amount of nitro-tyrosine-tripeptide to be almost six-fold lower
than that of nitro-phenylalanine-tripeptide. The results obtained
by 15N NMR were validated by high resolution mass spectrometry
(HRMS). The HRMS spectra revealed the presence of peaks
corresponding to H-Gly-Phe(15NO2)-Gly-OH (MNa+calculated
348.0932, found 348.0932), as well as H-Gly-Tyr(15 NO2)-Gly-OH
(MNa+calculated 364.0882, found 364.0888). The intensities of
the signals assigned to the nitration products, especially to H-Gly-
Tyr (NO 2)-Gly-OH, were much lower than those of H-Gly-Tyr-
Gly-OH and H-Gly-Phe(15NO2)-Gly-OH.
The NMR data fornitrated H-Gly-Trp-Gly-OHpeptide demon-
strated only traces of nitrated forms as judged from 15N (369.0
ppm) and 1H (8.07 ppm) chemical shifts (not shown). The strongest
cross peak in 15N-1H HMBC spectrum showed coupling between
positions 400.7 ppm 15N and 1.66 ppm 1H of the spectrum but
the chemical shift of the 1H resonance (singlet resonance) can
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Table 1 Experimental and calculated 15N NMR chemical shifts of nitrated side chains of selected amino acid moieties
Residue
dexp15 N (experimental; rel.
to NH3(lq)) [ppm]a
dexp1H (experimental; H
adjacent to N) [ppm]
dcal15 N (calculated; rel. to
NH3(lq)) [ppm]bDdc[ppm]
Nitro-product to
substrate ratiog
o-NO2Phe 382.8 — —
m-NO2Phe 389.7 — —
p-NO2Phe 389.7 -18.5 1 : 28.8
371.2; 374.1d8.09; 7.88d1 : 156.3d
2-NO2Trp 394.1e2.78e400.3 — —
4-NO2Trp 360.0e1.70e381.9 — —
5-NO2Trp 400.7e1.66e388.2 — —
6-NO2Trp 388.9 — —
7-NO2Trp 386.7 — —
7.84 (s, H-2)
3-NO2Tyr 373.4 7.37 (dd, H-6) 389.3 -15.9 1 : 4.4
6.93 (d, H-5)
2-NO2-His N1-H — 408.0
5-NO2-His N1-H 396.6
2-NO2-His N3-H 408.0
5-NO2-His N3-H 403.6
S-NO2Cys 8.38 (s)f370.0 — —
O-NO2Ser 8.39 (s)f418.2 — —
O-NO2Thr — 419.8
N-w-NO2Arg — 429.1
N-e-NO2Arg — 416.9
N-NO2Lys 8.39 (s)f407.7 —
aChemical shifts for nitrated tripeptides. bCalculated chemical shifts for truncated nitrated amino acids (Fig. 3). cDd=dexp15N-dcal 15N. dNO2-Tyr (see
Results and discussion). eSee Results and discussion. fSide chain oxidation product. gCalculated from 1H NMR spectrum.
not be associated with aromatic nitro-forms. It appears that the
above mentioned singlet resonance reflects a tryptophan ring-
cleavage product that contains an aliphatic region with 15N atom.12
Similar results, with only traces of nitro-form noticed (not shown),
were obtained when post reaction mixture containing H-Gly-His-
Gly-OH peptide was monitored. Moreover, the 15N-1HHMBC
spectrum of the nitrated H-Gly-His-Gly-OH sample did not show
any noticeable peaks corresponding to the 15Nand1H shifts of
nitro-histidine.
The NMR spectra of nitrated serine and lysine peptides showed
only traces of new forms. The 1H NMR spectra of H-Gly-Cys-
Gly-OH, H-Gly-Ser-Gly-OH and H-Gly-Lys-Gly-OH peptides
contained small resonances at 8.4 ppm, suggesting oxidation of
small percentage of peptides side chain heteroatoms to produce
aldehydes. The 1H spectra of nitrated threonine-containing peptide
also showed traces of O-nitro-threonine moiety (CH–O–NO2),
with its 1H resonance at 5.12 ppm and 15N resonance at 535.5 ppm
(not shown).
1H NMR spectrum of nitrated H-Gly-Cys-Gly-OH showed
almost all peptide used to be converted to various products.
The literature suggests that the reaction of cysteine moiety
with peroxynitrite may produce various amounts of disulfide
(RSSR), sulfenic acid (RSOH), sulfinic acid (RSO2H), sulfonic
acid (RSO3H), nitrosocysteine (RSNO), nitro-cysteine (RSNO2)
and various radicals.12 Although the existence of RSNO and
RSNO2, containing 15N-nitrogen atom originating from perox-
ynitrite, should be easy to confirm in 1D and 2D NMR spectra,
it was not apparent. Consequently, the new products found in
the 1H NMR spectrum appear to be mainly disulfide, as well as
sulfenic acid, sulfinic acid and sulfonic acid. The mass spectro-
metric analysis confirmed the presence of sulphonic acid in the
sample.
NMR analyses of nitrated TS
The nitration of protein conducted under acidic conditions (pH
2–5) resulted in the precipitation of protein and consequently
very low concentration of soluble 15N-nitro-forms that could be
analyzed by NMR method. The best results were achieved at
pD of 7.2–7.4; when in NMR tube no presence of visible solid
residues was apparent during the reaction and measurements. The
chemical shifts for the strongest peak found in 15N-1HHMBC
spectrum (7.67 ppm on the H-axis and 374.9 ppm on the 15N axis)
of nitrated protein (not shown) were in a very good agreement with
our data for nitrated H-Gly-Tyr-Gly-OH peptide (vide supra). Due
to relatively low concentrations of protein and very low yields of
nitration process, it was impossible to confirm identity of other
nitrated moieties.
MS analyses of nitrated TS
To localize the in vitro modifications of TS, nitrated human and C.
elegans proteins underwent proteolytic digestion and the resulting
peptides were analyzed by ESI. For each of the two proteins
several TS-derived peptides were found to contain nitro-tyrosine
(Table 2) and oxidized cysteine (Table 3) residues. It should be
mentioned that although sequences of several peptides listed
in Table 2 contained, besides tyrosine, also histidine residues, a
possibility of their nitration appears negligible. The nitration of
aromatic compounds is performed by the electrophilic aromatic
substitution mechanism. Susceptibility of tyrosine side chain for
electrophilic substitution is very high, in particular in its phenolate
form at pH >7. In contrast to this, the electron density of the
imidazole ring of histidine is lower. Consequently, the electrophilic
substitution of histidine residue is much slower and requires
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Fig. 4 Aromatic regions of H-Gly-Tyr-Gly-OH peptide (top) and post-reaction mixture of peptide 15N-nitration (bottom). Asterisks indicate aromatic
hydrogens of 15N-nitro-tyrosine moiety.
Fig. 5 The fragment of 15N-1H HMBC spectrum of the nitrated H-Gly–
Tyr-Gly-OH peptide showing 15N-H(Tyr) coupling. The largest peak on
15N axis belongs to NO3-anion.
higher pH than that of tyrosine. Therefore it appears hardly
possible that in the presence of very reactive phenolate side chain
the imidazole ring of histidine would be modified. In accord,
ESI-MS analysis revealed only one nitration site in the 255–261
fragment of human thymidylate synthase, containing one tyrosine
and two histidine residues (Fig. 6). Moreover, low susceptibility
to nitration of histidine vs. tyrosine was shown also by the results
of NMR analyses of nitrated model compounds (vide supra).
While the non-nitrated counterparts of several nitrated peptides
could be identified, in most cases their proportion was probably
too low to allow unambiguous recognition.
Following nitration at pH 7.5 with 20 mM H2O2, tyrosine
modification to nitro-tyrosine was found at positions 33, 65,
135, 213, 230, 258 and 301 in human TS (Fig. 6 and 7), and
34, 66, 137, 148 and 232 (homologous to human 33, 65, 135,
146 and 230, respectively) in the C. elegans protein. Interestingly,
when 50 mM H2O2was applied with C. elegans TS, a different
nitration profile, including positions 66, 137, 148, 204, 215 and
260 (homologous to human 65, 135, 146, 202, 213 and 258,
respectively), was apparent. Of note is a tendency for homologous
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Fig. 6 The ESI-MS spectrum of the chymotryptic products of human TS. The expanded part of spectrum corresponds to the fragment 255–261
containing the nitrated tyrosine at position 258. The monoisotopic molecular mass of the presented fragment was compared with one simulated basing
on the molecular formula.
Fig. 7 Ribbon representation of monomer A from the crystal structure
of human thymidylate synthase with bound dUMP and Tomudex (PDB
code: 1I00). Tyrosine residues are shown as sticks, labelled with sequence
numbers and marked either lighter or darker depending on whether a
residue has been found to be nitrated or not, respectively. dUMP and
Tomudex are shown as sticks and marked light.
tyrosine residues in both TS sequences to undergo nitration (Table
2). Considering localization of nitrated tyrosine residues in human
TS, those at positions 135, 230 and 258, being closest to the
active center (Fig. 7), were of particular interest, in view of the
influence of nitration on the catalytic potency (Fig. 2). It should
be mentioned that at the conditions used to nitrate the human
enzyme (pH 7.5, 20 mM H2O2) the only cysteine residue to undergo
modification was Cys210, with catalytically critical Cys195 remaining
apparently unmodified. In C. elegans, the homologous catalytic
cysteine residue TS (Cys197) underwent modification only at 50
mM H2O2, in accord with the dependence presented in Fig. 2,
and cysteine residues at positions 67, 151 and 243, oxidized at
20 mM H2O2, correspond to human TS homologous amino acid
residues different from cysteine (Ser66,Met
149 and Thr241,respec-
tively).
In order to asses a potential of nitration of different tyrosine
residues to affect TS catalytic potency, a parallel molecular
modeling study (using the molecular dynamics method, followed
by post-processing of the resulting trajectories) has been per-
formed. The simulations were based on the crystal structure of the
ternary complex of human thymidylate synthase with dUMP and
Tomudex (PDB accession code 1I00), with the Tomudex molecule
replaced by the molecule of tetrahydrofolate (close analogue of
methylenetetrahydrofolate), according to the previously described
superimposition.13 Initial results indicated nitration in human TS
of either of the four residues, Tyr33,Tyr
135,Tyr
213 and Tyr258,to
differently influence the binding alignment between the substrate,
dUMP, and cofactor, THF, in the enzyme active site. The impact
ranges from (i) a strong misalignment that is likely to significantly
reduce the catalytic activity of TS (nitration on Tyr135) to (ii)
moderate deviations from the native alignment (nitrations on
Tyr 33 and Tyr258), and (iii) the lack of deviation from the native
alignment (nitration on Tyr213)thatsuggestspreservationof
TS native catalytic activity.14 Considering the apparent strong
influence of Tyr135 nitration on the substrates alignment, of
interest is that mutation (Y94F) of the corresponding residue
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Table 2 Mass spectrometric determination of tyrosine residues modification of –NO2type in human and C. elegans recombinant thymidylate synthase
proteins nitrated in vitro at pH 7.5. Purified recombinant protein, following nitration, was analyzed by ESI-MS after proteolytic digestion. The peptides
listed encompass those resulting from digestion by trypsin, as well as chymotrypsin
Sequence of the modified peptide Modification site
Human TS protein nitrated at 20 mM H2O2
A228SYALL233 Ty r230
N205SELSCQLYQRSG217 Ty r213
F117LDSLGFSTREEGDLGPVYGFQW139 Tyr135
A255HIYLNH261 Ty r258
A22EPRPPHGELQYLGQIQHILRCGV45 Tyr33
T251LGDAHIY258 Tyr 258
S209CQLYQRSGDMGLGVPF225 Tyr213
K292AEDFQIEGYNPHPTIKM309 Ty r301
G60MQARYSLRD69 Tyr65
C. elegans TSproteinnitratedat20mMH
2O2-
K33YLKQVE39 Tyr 34 (Tyr33)a
L133GPVY137 GFQW141 Tyr137 (Tyr135)
F139QWRHFGAKYVDCHTDY155 Tyr 148;Tyr
155
G134PVYGFQWRHF144 Tyr 137 (Tyr135)
M221GLGVPFNLASYGL234 Tyr232 (Tyr230)
E274PYAFPK280 Ty r276
G61MQSKYCLRNG71 Ty r66 (Tyr65)
C. elegans TSproteinnitratedat50mMH
2O2
G61MQSKYCLRNG71 Ty r66 (Tyr65)
Q40ILREGTRRDDRTGTGTISIFGMQSKYCLRNGTIPLLTTKRV81 Tyr 66 (Tyr65)
G190QMVLPPCHTMCQFY204 Ty r204
T126SREEGDLGPVYGFQW141 Tyr 137 (Tyr135)
A146KYVDCHTD154 Ty r148
Y204VDNGELSCQLYQRSGDMG222 Ty r204 ;Tyr
215 (Tyr213)
G61MQSKYCLRNGTIPLLTTKRV81 Tyr66 (Tyr65)
T56ISIFGMQSKYCL68 Ty r66 (Tyr65)
T126SREEGDLGPVYGFQW141 Tyr 137 (Tyr135)
V251HTLGDAHVY260 Tyr 260 (Tyr258)
aHomologous human TS site, if also modified, is presented in parentheses.
in E. coli TS caused an apparent weakening of dUMP binding
and associated enhancement of dUMP release, resulting in both
substrates (dUMP and meTHF) interacting in a random binding
sequence.15
In view of the recently presented concept of hydrogen bond
bridges playing an important role in the reaction of protein
tyrosine nitration,16 it was of interest to extend our molecular
modeling studies to analyze how the results of nitration of
tyrosine residues in TS protein conformed to that concept.
The results were unequivocal. While the distance between the
nitrated tyrosine hydroxyl and the closest acidic/basic amino
acid side chain heavy atom matches always satisfactorily the
distance required for the nitrating species to form hydrogen bond
bridge connecting the tyrosine and corresponding charged amino
acid,16 the corresponding distances measured to heteroatoms of
the dissociable groups of the same amino acids are noticeably
different from those suggested to be optimal,16 and in accord,
orientations between those dissociable groups and the nitrated
tyrosine hydroxyls appear to be in most cases incorrect for forming
geometrically reasonable hydrogen bond bridges. However, the
latter statement should be taken with caution, as our molecular
modeling was performed for the systems that had already un-
dergone tyrosine nitration, hardly allowing evaluation of the in-
termediate conformational states occurring during nitration reac-
tions.
Conclusions
The present study suggests that thymidylate synthase protein,
expressed endogenously in normal and tumour (calf thymus and
L1210 cells) tissues undergoes nitration in vivo. The modification
may influence properties of the enzyme, as chemical reaction with
peroxynitrite (ONOO-) produced in situ at pH 7.5 of human,
mouse and C. elegans recombinant TS proteins, resulting primarily
in nitration of tyrosine residues, as confirmed by NMR and MS,
distinctly lowers the catalytic potency reflected by the Vmaxapp value.
Experimental
Materials
Tripeptides of general formula H-Gly-X-Gly-OH where X =Phe,
Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly were purchased from
Lipopharm (Poland).
Thymidylate synthase preparation
The endogenous enzyme proteins from parental and FdUrd-
resistant mouse leukemia L1210 cells,17 and calf thymus,18 were
purified as previously described. Ceanorhabditis elegans19 and
mouse20 TS coding regions were cloned into pPIGDM4+stop
vector and expressed as HisTag-free proteins in BL21(DE3)
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Table 3 Mass spectrometric determination of cysteine residues modification of –O type in human and C. elegans recombinant thymidylate synthase
proteins nitrated in vitro at pH 7.5. Purified recombinant protein, following nitration, was analyzed by ESI-MS after proteolytic digestion. The peptides
listed encompass those resulting from digestion by trypsin, as well as chymotrypsina
Sequence of the modified peptide Modification site
Human TS protein nitrated at 20 mM H2O2
N205SELSCQLYQRSG217 Cys210
C. elegans TSproteinnitratedat20mMH
2O2
G61MQSKYCLRNG71 Cys67
F144GAKYVDCHTDYSG157 Cys151
Q140WRHFGAKYVDCH152 Cys151
R142HFGAKYVDCHTDYSGQGVDQL163 Cys151
M238IAKVCGLKPGTLVH252 Cys243
C. elegans TSproteinnitratedat50mMH
2O2
G61MQSKYCLRNG71 Cys67
Q40ILREGTRRDDRTGTGTISIFGMQSKYCLRNGTIPLLTTKRV81 Cys67
G190QMVLPPCHTMCQFY204 Cys201
A146KYVDCHTD154 Cys151
K171EQPDSRRIIMSAWNPSDLGQMVLPPCHTMCQFYVDNGE209 Cys197;Cys
201
G190QMVLPPCHTMCQFYVDNGELSCQL214 Cys197 ;Cys
201;Cys
212 (Cys210)
aHomologous human TS site, if also modified, is presented in parentheses.
or thymidylate synthase-deficient TX61-(a kind gift from Dr
W. S. Dallas) E. coli strain, respectively. Human TS coding
regions21 were subcloned into pET28a vector and expressed as
HisTag-containing proteins in E. coli BL21(DE3) strain. HisTag
containing proteins were purified on NiNTA His-Bind resin
(Novagen) according to manufacturer protocol, and HisTag-free
proteins were purified as previously described.22 Phosphatase
inhibitors (50 mM NaF, 5 mM Na-pyrophosphate, 0.2 mM
EGTA, 0.2 mM EDTA and 2 mM Na3VO 4) were present in the
purification buffers. Each purified TS preparation was separated
from phosphorylated fraction according to Wolschin et al.,23
using metal oxide/hydroxide affinity chromatography on Al(OH)3
beads. The enzyme activity was measured and kinetic parameters
of the enzyme-catalyzed reaction were determined as previously
described.24
Thymidylate synthase tyrosine in vitro nitration
The reaction was performed at 4 C in the presence of 20 mM
dUMP (stabilization in the absence of 2-mercaptoethanol) in a
reaction mixture containing 200 mM Na/K phosphate buffer
pH 7.5, equimolar concentration of NaHCO3and H2O2(5–
70 mM), NaNO2at concentration by 5% exceeding the latter
(5.25–73.5 mM) and the enzyme (5 mMdimer).Tostartthe
reaction, H2O2was added, the sample mixed 30 s and next
incubated 5 min. While nitro-tyrosine content was determined
spectrophotometrically,25 to the remaining reaction mixture 2-
mercaptoethanol (20 mM) was added, followed by either protein
precipitation with 10% (w/v) TCA or sample dilution (300-
fold) with 50 mM Na/K phosphate buffer pH 7.5, containing
0.1% Triton X-100 and 10 mM 2-mercaptoethanol. The diluted
preparation preserved TS activity for at least 2 h, allowing enzyme
properties to be studied. To the control reaction mixture TS was
added after mixing and incubating the remaining components, in
order to inactivate the produced peroxynitrite.
Immunoblotting
Previously described method was used,13 with anti-tyrosine an-
tibody from Sigma–Aldrich (Cat. No. NO409) and anti-TS
antibody.19
Peptide nitration
Peptides of the general formula H-Gly-X-Gly (where X =Phe, Thr,
Trp, Lys, Cys, His, Ser, Arg, Gly) were nitrated in deuterium oxide
system using sealed 5 mm NMR tubes. Each tripeptide (8 mmol in
0.1 ml of deuterium oxide) was mixed with solutions containing
(i) 76 mmol H2O2in 0.1 ml of D2O, (ii) 80 mmol sodium 15N-nitrite
in 0.1 ml of D2O and (iii) sodium bicarbonate (80 mmol in 0.1 ml
D2O), followed by addition of 3.3 ml of conc. sulfuric acid in 0.1 ml
of D2O and additional D2O to the final volume of 0.6 ml (resulting
pD of 1.9).
NMR analyses
All NMR spectra were obtained with Bruker Avance spectrometer
operating in the quadrature mode at 500.13 MHz for 1H and 50.69
MHz for 15N nuclei. The residual peaks of deuterated solvents
were used as internal standards in 1H NMR method. 15NNMR
spectra were recorded at 277 K both with and without proton
decoupling. All 15N chemical shifts presented in this work are
related to liquid ammonia (0.0 ppm). The internal standard used
in 15N NMR was sodium nitrite (609.6 ppm rel. to liquid NH3)
and sodium nitrate (376.5 ppm rel. to liquid NH3). All samples
were analyzed using the gradient-enhanced 1H-15N Heteronuclear
Multiple Bond Correlation (HMBC) approach. The 1HNMR
spectra were obtained with the use of the HDO suppression
method. All buffer solutions used for NMR spectroscopy were
based on deuterium oxide of ‘100%D’ purity (Armar Chemicals
AG, Ge rm any).
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Calculations
The theoretical calculations have been performed with the density
functional B3LYP/aug-cc-pVTZ method. To save computational
time, the structures of amino acids were truncated by removing
some groups of atoms remote from the nitrated regions (Fig. 3).
Where it was possible, the calculations for full nitro-tripeptide
(GCG-NO2) were performed for comparison with the truncated
model. The optimal geometries were obtained and confirmed with
positive harmonic frequencies, then NMR shielding values for
models of nitroamino-derivatives of acids were calculated. All the
calculations were performed with the Gaussian G03 (rev. C.02)
suite of programs.
Mass spectrometry analysis
Enzymatic hydrolysis. A sample of modified protein (1 mg)
was dissolved in water (100 ml). The obtained solution (10 ml)
was diluted with 0.03 M NH4HCO3(50 ml). After adding the
0.1% water solution of the proteolytic enzyme (5 ml; trypsin or
chymotrypsin) the mixture was incubated at r.t. for 12 h. The
products of the hydrolysis were absorbed on OMIX C4 100 ml
pipette tips (Varian). The tip was washed 10 times with water
(100 ml), than the peptides formed by proteolysis of modified
protein were eluted with 60% water solution of acetonitrile
(100 ml).
Mass spectrometry. The enzymatic digest was analyzed on
high resolution ESI-FT mass spectrometer (Apex-Ultra Qe 7T;
Bruker Daltonics; Germany) equipped with an electrospray ion-
ization (ESI) source. The instrument was operated both in the
positive and negative ion mode and calibrated with the Tunemix
mixture (Bruker Daltonics). The mass accuracy was better than
5 ppm. The instrumental parameters were as follows: scan range,
300–2500 m/z; drying gas, nitrogen; temperature of drying gas,
200 C; potential between spray needle and orifice, set at 4.5 kV;
source accumulation time, 0.5 s; and ion accumulation time, 0.5 s.
The analyzed solution was infused directly to the ion source at a
flow rate 3 mlmin
-1. Each spectrum is an average of more than 100
individual scans.
Data analysis. The analysis of spectra was performed using the
SNAP algorithm (Data Analysis, Bruker). The generated mass
list, including m/zratio, monoisotopic mass and z,wasfurther
analyzed using Excel spreadsheet basing on the following assump-
tions: (i) The cleavage sites for trypsin: [K,R] and for chymotrypsin
[Y,W,F,L,M,H,N,G,I,V,E,D]; (ii) accepted chemical modifications
nitration of tyrosine and oxidation of the methionine and cysteine;
(iii) Accepted error max. 10 ppm; (iv) Peptide length max. 20
amino acid residues; (v) Only protonated peptides were accepted,
the metal adducts (Na, K, Ca) were neglected.
Acknowledgements
Supported by the Ministry of Science and Higher Education
(grant numbers N401 0612 33 and N N401 0240 36). Stimulating
discussions from COST Action CM1001 are acknowledged.
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Models for exploring tyrosine nitration in proteins have been created based on 3D structural features of 20 proteins for which high-resolution X-ray crystallographic or NMR data are available and for which nitration of 35 total tyrosines has been experimentally proven under oxidative stress. Factors suggested in previous work to enhance nitration were examined with quantitative structural descriptors. The role of neighboring acidic and basic residues is complex: for the majority of tyrosines that are nitrated the distance to the heteroatom of the closest charged side chain corresponds to the distance needed for suspected nitrating species to form hydrogen bond bridges between the tyrosine and that charged amino acid. This suggests that such bridges play a very important role in tyrosine nitration. Nitration is generally hindered for tyrosines that are buried and for those tyrosines for which there is insufficient space for the nitro group. For in vitro nitration, closed environments with nearby heteroatoms or unsaturated centers that can stabilize radicals are somewhat favored. Four quantitative structure-based models, depending on the conditions of nitration, have been developed for predicting site-specific tyrosine nitration. The best model, relevant for both in vitro and in vivo cases, predicts 30 of 35 tyrosine nitrations (positive predictive value) and has a sensitivity of 60/71 (11 false positives).
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Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.
Article
The role of the phosphate moiety of dUMP, and some analogues, in their interaction with mammalian thymidylate synthase, has been investigated. Substrate and inhibitor activities, and the pH-dependence of these activities, of dUMP and 5-FdUMP, as well as analogues with modified phosphate groups, were compared. The methyl ester of dUMP was neither a substrate nor an inhibitor. By contrast, the methyl ester of 5-FdUMP was a slow-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells, with Ki values in the micromolar range. Both 5-FdUrd and the newly synthesized 5'-methylphosphonate of 5-FdUrd were also slow-binding inhibitors of the Ehrlich carcinoma enzyme, but with Ki values in the millimolar range. The interaction of dUMP, 5-FdUMP, and the methyl ester of the latter decreased with increase in pH, whereas that of the 5'-methyl-phosphonate of 5-FdUrd remained unchanged. The results are discussed in relation to the role of the phosphate hydroxyls of dUMP in binding to the enzyme. 5-FdUMP and its analogues exhibited differing interactions with two binding sites on the enzyme molecule, consistent with cooperativity of binding. A convenient procedure is described for the synthesis of 5-fluoro-2'-deoxyuridine-5'-methylphosphonate, applicable also to the preparation of other 5'-methylphosphonate analogues.
Article
1.1. Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells.2.2. The enzyme is a dimer composed of 35,000 mol. wt monomers.3.3. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP.4.4. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34† C, reflecting a change of rate-limiting step in dTMP formation.5.5. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
Article
Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine ring of the substrate, dUMP, and the pterine ring of the cofactor analogue, THF, in the active site of the phosphorylated enzyme. The ED analysis indicates changes in the behavior of collective motions in the phosphorylated enzyme, suggesting that the formation of the closed ternary complex is hindered. Computed free energies, in agreement with structural analysis, predict that the binding of dUMP and THF to hTS is favored in the native compared to phosphorylated state of the enzyme. The paper describes at the structural level how phosphorylation at the distant site influences the ligand binding. We propose that the 'phosphorylation effect' is transmitted from the outside loop of Ser 124 into the active site via a subtle mechanism initiated by the long-range electrostatic repulsion between the phosphate groups of dUMP and Ser124. The mechanism can be described in terms of the interplay between the two groups of amino acids: the link (residues 125-134) and the patch (residues 189-192), resulting in the change of orientation of the pyrimidine ring of dUMP, which, in turn, prevents the correct alignment between the latter ring and the pterin ring of THF.
Article
Sulfhydryl-blocked thymidylate synthetase (EC 2.1.1.4.5) is rapidly inactivated by low concentrations of tetranitromethane. This reagent first nitrates two non-essential tyrosines per dimeric enzyme molecule followeed by two essential tyrosines with no oxidation of sulfhydryl groups. dUMP affords significant protection against inactivation. These results suggest that essential tyrosyl residues are present in the active sites of the enzyme.
Article
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.
Article
Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.
Article
This chapter discusses the detection and quantitation methods of nitrotyrosine residues in proteins. Nitrotyrosine is detected in human diseases associated with oxidative stress and is visualized using immunological techniques in atherosclerotic plaques of human coronary vessels, in lungs of infants with acute lung injury and sepsis, and in adult respiratory distress syndrome (ARDS). High-performance liquid chromatography (HPLC) analysis is used to detect nitrotyrosine in synovial fluid from patients with rheumatoid arthritis. Peroxynitrite can be synthesized from sodium nitrite and acidified hydrogen peroxide. Selective tyrosine nitration can be accomplished by titrating protein with tetranitromethane (TNM) at neutral or alkaline conditions (pH 7–8). TNM is a potent carcinogen, which must be handled carefully. The residual TNM and trinitromethane must be removed prior to nitrotyrosine quantitation. Nitrotyrosine is essentially nonfluorescent whereas aminotyrosine is highly fluorescent and has a characteristic emission spectrum. Thus, fluorescent detection of aminotyrosine can be used as an alternative to direct detection of nitrotyrosine. Quantitation of nitrotyrosine using the solid-phase immunoradiochemical method has the advantage of high sensitivity and does not require sample manipulation.