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The Journal of Neuroscience, December 1, 1999, 19(23):10289-10294
Peroxynitrite Inactivation of Tyrosine Hydroxylase: Mediation by
Sulfhydryl Oxidation, not Tyrosine Nitration
Donald M.
Kuhn1, 2,
Cheryl W.
Aretha1, and
Timothy J.
Geddes1
1 Department of Psychiatry and Behavioral Neurosciences
and 2 Center for Molecular Medicine and Genetics, Wayne
State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme
in the biosynthesis of dopamine (DA). TH activity is significantly diminished in Parkinson's disease (PD) and by the neurotoxic
amphetamines, thereby accentuating the reductions in DA associated with
these conditions. Reactive oxygen and nitrogen species have been
implicated in the damage to DA neurons seen in PD and in reaction to
amphetamine drugs of abuse, so we investigated the hypothesis that
peroxynitrite (ONOO ) could interfere with TH
catalytic function. ONOO caused a
concentration-dependent inactivation of TH. The inactivation was
associated with tyrosine nitration (maximum of four tyrosine residues
nitrated per TH monomer) and extensive sulfhydryl oxidation. Tetranitromethane, which causes sulfhydryl oxidation at pH 6 and 8 but
which nitrates tyrosines only at pH 8, inactivated TH equally at either
pH. Bicarbonate protected TH from ONOO-induced
inactivation and sulfhydryl oxidation but increased significantly tyrosine nitration. PNU-101033 blocked
ONOO-induced tyrosine nitration in TH but could
not prevent enzyme inactivation or sulfhydryl oxidation. Together,
these results indicate that the inactivation of TH by
ONOO is mediated by sulfhydryl oxidation. The
coincident nitration of tyrosine residues appears to exert little
influence over TH catalytic function.
Key words:
peroxynitrite; tyrosine hydroxylase; tyrosine nitration; sulfhydryl oxidation; dopamine; Parkinson's disease; neurotoxic
amphetamines
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INTRODUCTION |
Tyrosine hydroxylase (TH) is the
initial and rate-limiting enzyme in the biosynthesis of the
neurotransmitter dopamine (DA). DA modulates a variety of physiological
and behavioral processes, and alterations in its function have been
implicated in numerous psychiatric and neurological illnesses. The
neurotoxic amphetamines (e.g., methamphetamine) cause both short- and
long-term reductions in TH activity (O'Callaghan and Miller, 1994 ;
Albers and Sonsalla, 1995; Gibb et al., 1997; Haughey et al., 1999),
and it is likely that chronic abuse of these drugs could have adverse
effects on those processes mediated by DA. TH function and DA levels
are reduced substantially in Parkinson's disease (PD), but the extent of their reduction is greater than the loss of DA neurons that occurs
in this disorder (Hornykiewicz and Kish, 1987; Pakkenburg et al.,
1991). Therefore, it appears that TH activity is actively suppressed,
even in the face of ongoing DA cell loss. Using the DA neurotoxin
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to model the loss
of DA neurons characteristic of PD, Ara et al. (1998) demonstrated
recently that TH was inhibited by a peroxynitrite (ONOO)-mediated nitration of intrinsic
tyrosine residues.
ONOO is a highly reactive species
produced by the near-diffusion limited reaction of nitric oxide with
superoxide (Huie and Padjama, 1993) and is itself cytotoxic (Beckman
and Koppenol, 1996). ONOO has been
implicated as a causative factor in DA neuronal damage seen in PD
(Schulz et al., 1995; Hantraye et al., 1996; Przedborski et al.,
1996; Good et al., 1998), as well as after administration of
neurotoxic amphetamines (Cadet et al., 1994; Di Monte et al., 1996;
Sheng et al., 1996; Itzhak et al., 1998; Tsao et al., 1998; Imam et
al., 1999). In addition to nitrating tyrosine residues in proteins,
ONOO is also known to be a powerful
sulfhydryl oxidant (Radi et al., 1991; Quijano et al., 1997). In view
of the importance of identifying ONOO as
a possible mediator of reduced TH activity under conditions known to
damage DA neurons and considering the complexity of its actions on
proteins, the aim of the present study was to assess the relative
contribution of sulfhydryl and tyrosine modification by
ONOO to the inactivation of TH.
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MATERIALS AND METHODS |
tk;2Materials. Tryptophan, dithiothreitol, superoxide
dismutase, diethylenetriamine penta acetic acid (DTPA),
5,5'-dithiobis-2-nitrobenzoic acid (DTNB),
N-acetyl-imidazole, methionine, cysteine, DMSO, dithionite, glutathione, glutathione-agarose, sodium arsenite, sodium bicarbonate, sodium borohydride, and uric acid were obtained from Sigma (St. Louis,
MO). Catalase and a monoclonal antibody against TH were products
of Boehringer Mannheim (Indianapolis, IN). Tetranitromethane (TNM) was
purchased from Aldrich (Milwaukee, WI).
Isopropyl- -thiogalactopyranoside was obtained from Gold
Biotechnologies. Thrombin and pGEX vectors were obtained from Amersham
Pharmacia Biotech (Arlington Heights, IL). Tetrahydrobiopterin was
purchased from Dr. Shircks Laboratories (Jona, Switzerland). A
monoclonal antibody against nitrotyrosine was purchased from Cayman
Chemical Company (Ann Arbor, MI), and horseradish peroxidase-linked
goat anti-mouse IgGs were products of Cappel (West Chester, PA).
Enhanced chemiluminescence reagents were products of DuPont NEN
(Boston, MA), and Bio-Max MR film was from Eastman Kodak (Rochester,
NY). Restriction endonucleases, T4 ligase, and T4 kinase were products
of New England Biolabs (Beverly, MA). All other reagents were obtained
from commercial sources in the highest possible qualities.
Preparation of TH and treatment with
ONOO. TH was cloned by reverse
transcription-PCR and expressed as a glutathione
S-transferase fusion protein as described previously for
tryptophan hydroxylase (D'Sa et al., 1996). Nucleotide sequencing
confirmed that full-length TH had been cloned accurately. The
recombinant protein was purified by glutathione-agarose affinity
chromatography, and the glutathione S-transferase fusion tag
was removed by thrombin cleavage, resulting in a highly purified TH
preparation (>95% pure). ONOO was
synthesized by the quenched-flow method of Beckman et al. (1994), and
its concentration was determined by the extinction coefficient
 302 = 1670 M1
cm1. The hydrogen peroxide contamination of
ONOO solutions was removed by manganese
dioxide chromatography and filtration (Beckman et al., 1994).
ONOO was added to TH with vigorous
mixing in 50 mM potassium phosphate buffer, pH
7.4, containing 100 µM DTPA, and incubations
were performed for 15 min at 30°C. The volume of
ONOO added to the enzyme samples was
always <1% (v/v) and did not influence pH. Protective agents, when
present, were added 5 min before ONOO.
The effects of bicarbonate on
ONOO-induced modifications of TH were
tested as described by Radi et al. (1999). Upon completion of
incubation with ONOO, samples were
diluted with an equal volume of 50 mM potassium phosphate, pH 6, and stored at 4°C. Residual TH activity was assayed according to the method of Lerner et al. (1978). Protein concentrations were determined as described by Bradford (1976).
Treatment of TH with TNM. TH was exposed to TNM (diluted in
ethanol) at either pH 6 or 8. TH was diluted into buffer at the appropriate pH (50 mM potassium phosphate at pH 6 or 8), and TNM was added and allowed to react with the protein for 15 min at 30°C. Enzyme samples were diluted with an equal volume of 50 mM potassium phosphate, pH 6, and assayed for
residual activity as described. TNM-modified TH was also tested for
sulfhydryl content and for tyrosine nitration as described below. The
control for TNM was an equivalent volume of ethanol.
Analysis of TH sulfhydryl and nitrotyrosine content. The
effect of ONOO or TNM on TH sulfhydryl
content was determined by the method of Ellman (1959). After treatment
with ONOO or TNM, an aliquot of enzyme
was removed and assayed for TH activity. The remaining sample (2-5
µM) was denatured with SDS (1% final concentration) and reacted with 200 µM DTNB at
room temperature for 90 min. Reactions were monitored as increases in
absorbance at 412 nm, and TH sulfhydryl content was determined by the
extinction coefficient 412 = 13,600 M1 cm1. The effect
of ONOO on TH nitrotyrosine content was
determined according to Crow and Ischiropoulos (1996).
ONOO was added to TH in 100 µM increments with vigorous mixing, and reactions were monitored as increases in absorbance at 430 nm. Nitrotyrosine content was determined by the extinction coefficient 430 = 4400 M1
cm1.
SDS-PAGE and Western blot analysis of TH. After treatment
with ONOO or TNM, TH was subjected to
SDS-PAGE on 10% gels according to Laemmli (1970). Proteins were
transferred to nitrocellulose, blocked in Tris-buffered saline
containing Tween 20 (0.1% v/v) and 5% nonfat dry milk, and probed
with a monoclonal antibody specific for nitrotyrosine. In some
experiments, blots were stripped and reprobed with a monoclonal
antibody specific for TH. After incubations with primary antibodies,
blots were incubated with goat anti-mouse secondary antibody conjugated
with horseradish peroxidase, and immunoreactive protein bands were
visualized with enhanced chemiluminescence.
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RESULTS |
Effect of ONOO on TH activity
ONOO caused a
concentration-dependent inactivation of TH, as shown in Figure
1. The IC50 for
inactivation was ~160 µM, and concentrations of
ONOO above 500 µM
inhibited TH >80%. Reverse-order addition of
ONOO to TH did not have an effect on
enzyme activity. Reagents known to interact directly with
ONOO, including glutathione, cysteine,
dithiothreitol, methionine, and uric acid, significantly protected TH
from inactivation. Catalase, superoxide dismutase, or dimethyl
sulfoxide, which scavenge hydrogen peroxide, superoxide, or hydroxyl
radical, respectively, did not protect TH from
ONOO-induced inactivation (data not
shown).
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Figure 1.
ONOO inhibits TH activity. TH
(20 µg) was incubated with the indicated concentrations of
ONOO ( ) or with decomposed
ONOO ( ) for 15 min at 30°C in 50 mM potassium phosphate buffer, pH 7.4, containing 100 µM DTPA. After treatment, enzyme samples were diluted 1:2
with buffer, and residual TH activity was determined. Each
symbol represents the mean ± SEM of four
independent experiments performed in duplicate, and data are expressed
as percentage control TH activity.
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Nitration of TH by ONOO
TH was not reactive with a monoclonal antibody specific for
nitrotyrosine until it was treated with
ONOO and, as shown in Figure
2A, the TH monomer
[molecular weight (MW) of 60 kDa] was increasingly nitrated by
ONOO up to a concentration of 0.5 mM. ONOO
concentrations above this appeared to diminish the amount of immunoreactivity associated with the TH monomer, and a second immunoreactive band appeared at a MW of ~120 kDa. When blots were stripped and reprobed with a monoclonal antibody specific for TH, both
the 60 and 120 kDa bands were found to be immunopositive for TH (Fig.
2B). Spectrophotometric measures of tyrosine
nitration in TH confirmed that ONOO
caused a maximal nitration of 3.8 tyrosines per TH monomer at a
cumulative concentration of 2.5 mM (data not
shown). These results suggest that ONOO
nitrates TH and causes the formation of TH dimers via dityrosine cross-linking.
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Figure 2.
ONOO nitrates tyrosine
residues in TH and causes the formation of dityrosines.
A, TH was treated with the indicated concentrations of
ONOO for 15 min at 30°C and exposed to SDS-PAGE
and immunoblotting with a monoclonal antibody specific for
nitrotyrosine (diluted 1:1000). B, Blots were stripped
and reprobed with a monoclonal antibody specific for TH (diluted
1:20,000). Immunoreactive proteins were visualized by enhanced
chemiluminescence with the use of goat anti-mouse secondary antibodies
conjugated to horseradish peroxidase. The positions of MW standards (in
kilodaltons) are illustrated to the right of the
blot.
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Effect of ONOO on TH sulfhydryl content
ONOO caused a
concentration-dependent loss of DTNB-reactive sulfhydryl groups in TH,
as shown in Figure 3. Untreated TH
contained seven sulfhydryls per monomer, in agreement with the number
of cysteine residues in the TH monomer as predicted from its nucleotide sequence (Grima et al., 1985). At the approximate
IC50 for inactivation of TH catalytic activity
(200 µM), ONOO reduced the
number of sulfhydryls by 50%. Indeed, Figure 3 shows that the loss of
TH catalytic activity paralleled the loss of sulfhydryl groups, and
these two measures were highly correlated (r2= 0.98; p < 0.05). The oxidized sulfhydryl groups of
ONOO-treated TH could not be reduced by
sodium arsenite or sodium borohydride, suggesting that they had been
oxidized beyond sulfenic acid (Radi et al., 1991) (data not shown).
Additionally, the sulfhydryl reagents DTNB (10 mM), o-iodosobenzoic acid (5 mM), and p-chloromercuribenzoic acid
(0.1 mM) caused complete inactivation of TH (data
not shown).
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Figure 3.
The effect of ONOO on TH
activity and sulfhydryl content. TH (120 µg) was reacted with the
indicated concentrations of ONOO for 15 min at
30°C. An aliquot of enzyme was removed for measures of residual TH
activity (), and the remaining enzyme was analyzed for sulfhydryl
reactivity ( ) with DTNB. Each symbol represents the
mean ± SEM of three to four independent experiments performed in
duplicate.
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Effect of TNM on TH
Because ONOO nitrates tyrosines and
oxidizes sulfhydryl groups in TH, additional experiments were needed to
determine the relative contribution of these modifications to enzyme
inactivation. TNM was useful in this regard because it is known to
oxidize sulfhydryls at pH 6 and 8, but it selectively nitrates tyrosine
residues at pH 8 (Sokolovsky et al., 1966; MacMillan-Crow et al.,
1998). Figure 4A shows
that TNM caused a concentration-dependent inactivation of TH, and this
inhibitory effect of TNM was the same at either pH 6 or 8. Figure
4A also shows that TNM caused the loss of
DTNB-reactive sulfhydryl groups from TH, and this effect occurred in a
pH-independent manner as well. The TNM-induced loss of TH catalytic
activity and sulfhydryl groups was highly correlated at both pH values (r2 = 0.95 for each pH;
p < 0.05). Figure 4B confirms that
the tyrosine nitrating properties of TNM are highly pH-dependent. TH is
minimally nitrated by TNM at pH 6 in concentrations well above those
that cause complete inactivation of enzyme activity (Fig.
4A). However, at pH 8, TNM causes a
concentration-dependent nitration of tyrosine residues in TH. The
extent of nitration of TH at pH 6 was estimated from pixel densities of
digitized scans of immunoblots to be <1% of the nitration at pH 8. TNM did not result in the formation of dityrosines at pH 8, consistent
with results with manganese superoxide dismutase (MacMillan-Crow et
al., 1998).
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Figure 4.
The effect of TNM on TH. A, TH was
treated with the indicated concentrations of TNM in 50 mM
potassium phosphate buffer containing 100 µM DTPA at pH 6 (,  ) or pH 8 ( , ) for 15 min at 30°C. An aliquot of the
enzyme was removed and analyzed for residual TH activity
(filled symbols), and the remaining enzyme was
analyzed for sulfhydryl reactivity (open symbols) with
DTNB. Data represent means ± SEM of three to four independent
experiments performed in duplicate. B, TH was treated
with the indicated concentrations of TNM at pH 6 or 8 and exposed to
SDS-PAGE and immunoblotting with a monoclonal antibody specific for
nitrotyrosine. Immunoreactive proteins were visualized by enhanced
chemiluminescence with the use of goat anti-mouse secondary antibodies
conjugated to horseradish peroxidase. TNM does not cause the formation
of dityrosines at pH 8, so the blot shows only the TH monomer at 60 kDa.
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Modulation of ONOO effects on TH by
bicarbonate and PNU-101033
Two additional experiments were performed in an attempt to align
the ONOO-induced inactivation of TH with
sulfhydryl oxidation or tyrosine nitration. First, bicarbonate was
tested for its effects because of the well known ability of
CO2 to react with
ONOO to produce the
ONOOCOO intermediate. This intermediate
is a more effective tyrosine nitrating agent and a less effective
sulfhydryl oxidant than ONOO (Lynar and
Hurst, 1995; Denicola et al., 1996; Gow et al., 1996; Uppu et al.,
1996; Zhang et al., 1997). The results in Table
1 indicate that bicarbonate (10 mM) significantly protected TH from ONOO-induced loss of catalytic activity
and sulfhydryl oxidation but caused a significant increase in nitration
of tyrosine residues. Second, PNU-101033 is known to block
ONOO-induced nitration of tyrosine
residues in proteins without interfering with sulfhydryl oxidation
(Rohn and Quinn, 1998). Table 1 also shows that PNU (100 µM) prevented neither the
ONOO-induced inactivation of TH nor the
loss of sulfhydryls, but it was very effective in blocking nitration of
tyrosine residues in TH.
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DISCUSSION |
TH is an important enzyme because it catalyzes the initial and
rate-limiting step in the biosynthesis of the neurotransmitter DA. The
clinical relevance of losses of TH activity is well documented in PD.
It is also known that the severity of PD symptoms is correlated with
the magnitude of DA (i.e., TH) reduction. Therefore, suppression of TH
activity, even in the face of ongoing neuronal degeneration, is a
contributing factor to the manifestation of PD. Reductions in DA
content and TH activity are also caused by neurotoxic doses of the
substituted amphetamines, such as methamphetamine and
3,4-methylenedioxymethamphetamine (O'Callaghan and Miller, 1994;
Albers and Sonsalla, 1995; Gibb et al., 1997; Haughey et al., 1999).
The mechanisms by which TH activity is suppressed in PD or by the
amphetamines are not known, but emphasis has been placed on reactive
oxygen species and reactive nitrogen species as possible mediators of
this inhibition (Van der Vliet et al., 1996; Jenner and Olanow, 1998;
Tsao et al., 1998).
An important step forward in identifying a possible mechanism of
TH inhibition was made recently with the demonstration that MPTP
reduces TH activity via ONOO-mediated
tyrosine nitration (Ara et al., 1998).
ONOO also inhibits
L-3,4-dihydroxyphenylalanine synthesis in PC12 cells
(Ischiropoulos et al., 1995). ONOO is
well known for its ability to nitrate tyrosine residues in proteins
(Ischiropoulos et al., 1992), and this effect has been applied as a
marker for ONOO action in ischemia,
Alzheimer's disease, amyotrophic lateral sclerosis, and autoimmune
disease (Beckman and Koppenol, 1996; Bruijn et al., 1997; Hensley et
al., 1998; Paris et al., 1998). ONOO-mediated tyrosine nitration has
even been implicated in the pathogenesis of PD (Schulz et al., 1995;
Hantraye et al., 1996; Przedborski et al., 1996; Good et al., 1998).
However, ONOO is also a powerful
sulfhydryl oxidant, and this effect may predominate over that of
tyrosine nitration when one considers the mechanisms by which proteins
can be altered by ONOO (Alvarez et al.,
1999). The role of sulfhydryl oxidation in TH catalytic function is not
known, but it can be inferred by analogy to tryptophan hydroxylase
(Kuhn et al., 1980; Kuhn and Arthur, 1997), a structurally and
functionally related monooxygenase, that TH activity is strongly
influenced by the redox status of its sulfhydryl groups. The aim of the
present studies was to assess the relative roles of sulfhydryl
oxidation and tyrosine nitration in the
ONOO-induced inactivation of TH.
Our results confirmed that ONOO is a
potent inhibitor of TH activity. The IC50 was
~160 µM. It has been suggested that the bolus addition
of 0.25 mM ONOO is
approximately equivalent to a steady-state level of 1.0 µM maintained for 7 min (Radi et al., 1991; Beckman et
al., 1994). Because ONOO can be formed
in vivo by the diffusion-limited reaction of nitric oxide
and superoxide anion, TH could well be a target for
ONOO in vivo, as suggested by
the case of MPTP (Ara et al., 1998). Various agents were tested for the
ability to protect TH from ONOO-induced
activation. Glutathione, dithiothreitol, cysteine, methionine, and uric
acid were very effective in this regard, consistent with their known
reactivities with ONOO (Halliwell et
al., 1999). It does not appear that the effect of
ONOO on TH activity is mediated by
radicals or reactive oxygen species because scavengers of hydroxy
radical (DMSO), hydrogen peroxide (catalase), or superoxide (superoxide
dismutase) did not protect the enzyme from inactivation. Finally, the
reverse-order addition of ONOO to TH did
not cause enzyme inhibition, strengthening the conclusion that
ONOO was the inactivating species.
ONOO caused a concentration-dependent
increase in the nitration of tyrosines in TH as measured by
immunoblotting. Spectrophotometric studies confirmed that
concentrations of ONOO causing 50%
inhibition of catalytic activity (~150 µM) resulted in
the nitration of ~0.7 tyrosine residues per TH monomer. A maximum of
3.8 tyrosines per TH monomer were nitrated at a cumulative ONOO concentration of 2.5 mM. In addition to causing tyrosine nitration of TH,
ONOO also caused dityrosine formation. A
second protein of 120 kDa was detected on immunoblots by the
nitrotyrosine antibody, and at the highest
ONOO concentrations tested (2 mM), this band was predominant. The 60 kDa protein (TH
monomer) and the higher molecular weight band were also immunoreactive
for TH. These results indicate that TH monomers were cross-linked into
dimers via dityrosine formation. It is well known that
ONOO can form dityrosines in proteins
through intermediate tyrosyl radicals (Anderson, 1966; Lehrer and
Fasman, 1967; Ischiropoulos and Al-Mehdi, 1995; MacMillan-Crow et al.,
1998; MacMillan-Crow and Thompson, 1999).
ONOO caused a concentration-dependent
loss of sulfhydryl groups from TH, and this effect was highly
correlated with the loss of enzyme activity. Untreated TH contained
seven sulfhydryls per monomer, consistent with the number predicted
from its nucleotide sequence (Grima et al., 1985). Concentrations of
ONOO causing 50% inhibition of TH
activity reduced the number of sulfhydryls from seven per monomer to
three to four, and concentrations that totally inhibited TH catalytic
activity reduced sulfhydryls to less than one per TH monomer. It
appears from these results that TH catalysis is influenced by the
status of its sulfhydryl groups, with loss of sulfhydryls being tightly
associated with loss of function. The sulfhydryl reagents DTNB,
o-iodosobenzoic acid, and p-chloromercuribenzoic
acid were capable of inactivating TH (data not shown). The fact that
sulfhydryl reagents can inactive TH without modifying tyrosine residues
establishes that sulfhydryl modification by
ONOO could play an important role in
inhibiting enzyme catalytic activity.
In an attempt to assess the relative contributions to TH inhibition
made by sulfhydryl oxidation and tyrosine nitration, we tested TNM for
its effects on TH. TNM was useful in this regard because it oxidizes
protein sulfhydryls in a pH-independent manner, but its tyrosine
nitrating properties are highly pH-dependent, occurring at pH 8 but not
at pH 6 (Sokolovsky et al., 1966; MacMillan-Crow et al., 1998). TNM
caused a concentration-dependent inactivation of TH, and this loss of
activity was highly correlated with the loss of sulfhydryl groups, as
was the case for ONOO. Both enzyme
inactivation and loss of sulfhydryls were pH-independent. TNM also
caused tyrosine nitration at pH 8 with very little nitration resulting
at pH 6. We predicted that if tyrosine nitration was mediating TH
activity, TNM would be more inhibitory at pH 8 versus pH 6. Therefore,
it is significant that TNM was no more inhibitory of enzyme activity
(or loss of sulfhydryl groups) at pH 8 compared with pH 6. These
results indicate that the tyrosine nitrating properties of TNM at pH 8 did not increase inactivation of TH compared with pH 6 and suggest that
sulfhydryl modification plays the predominant role in enzyme inhibition
caused by at least TNM.
In view of the importance of determining the role of tyrosine residues
in TH catalytic activity, two other reagents were tested for their
effects on ONOO-induced modification of
TH. First, bicarbonate (i.e., CO2) reacts readily
with ONOO to produce the
ONOOCOO intermediate, and this species
is known to be a more effective tyrosine nitrating species and a less
effective sulfhydryl oxidant than ONOO
(Denicola et al., 1996; Gow et al., 1996; Uppu et al., 1996; Zhang et
al., 1997; Alvarez et al., 1999). Bicarbonate protected TH from
ONOO-induced inactivation and loss of
sulfhydryl groups but significantly increased tyrosine nitration.
Second, PNU-101033 has been shown to prevent
ONOO-induced nitration without blocking
the sulfhydryl oxidizing effects of ONOO
(Rohn and Quinn, 1998). The results showed that, although this compound
provided only slight protection against
ONOO-induced inhibition of TH and loss
of sulfhydryl groups, it completely prevented tyrosine nitration in TH.
Together, the results with TNM, bicarbonate, and PNU-101033 do not
indicate an important role for tyrosine nitration in determining the
catalytic activity of TH.
The data of Ara et al. (1998) made the important observation that TH
activity can be modulated in vivo and in situ by
tyrosine nitration in response to MPTP administration and
ONOO, respectively. It would be
extremely difficult, if not impossible, to assess the status of TH
sulfhydryl groups under the same conditions. The present in
vitro experiments stress the importance of sulfhydryl groups in
determining TH catalytic activity and are hard to reconcile with the
results of Ara et al. (1998). Although our conclusions differ from
those of Ara et al. (1998) with regard to mechanism of inhibition of TH
by ONOO, it would be likely that
ONOO-induced nitration of tyrosines
in vivo could occur along with sulfhydryl oxidation as it
does in vitro Therefore, at a minimum, measures of TH
tyrosine nitration would provide valuable evidence for the action of
reactive nitrogen species in pathophysiological and drug-induced
conditions known to cause reductions in TH activity and DA deficits.
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FOOTNOTES |
Received July 16, 1999; revised Sept. 2, 1999; accepted Sept. 20, 1999.
This work was supported by National Institute on Drug Abuse Grant DA
10756 and grants from the Parkinson's Disease Foundation and the Joe
Young, Sr. Psychiatric Research Fund of the Department of Psychiatry
and Behavioral Neurosciences. We thank Dr. Philip Von Voigtlander of
Pharmacia & Upjohn, Inc. for the generous gift of PNU-101033.
Correspondence should be addressed to Donald M. Kuhn, Department of
Psychiatry and Behavioral Neurosciences, Wayne State University School
of Medicine, 2125 Scott Hall, 540 East Canfield, Detroit, MI 48201. E-mail: donald.kuhn{at}wayne.edu.
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ABSTRACT
INTRODUCTION
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