From the Department of Chemistry, University of Minnesota, Duluth, Minnesota 55812
Received for publication, August 21, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Biological systems that produce or are
exposed to nitric oxide (NO·) exhibit changes in the rate of
oxygen free radical production. Considering that mitochondria are the
main intracellular source of oxygen radicals, and based on the recently
documented production of NO· by intact mitochondria, we
investigated whether NO·, produced by the mitochondrial
nitric-oxide synthase, could affect the generation of oxygen radicals.
Toward this end, changes in H2O2
production by rat liver mitochondria were monitored at different rates
of endogenous NO· production. The observed changes in
H2O2 production indicated that NO·
affected the rate of oxygen radical production by modulating the rate
of O2 consumption at the cytochrome oxidase level. This mechanism was supported by these three experimental proofs: 1) the reciprocal correlation between H2O2
production and respiratory rates under different conditions of
NO· production; 2) the pattern of oxidized/reduced carriers in
the presence of NO·, which pointed to cytochrome oxidase as the
crossover point; and 3) the reversibility of these effects, evidenced
in the presence of oxymyoglobin, which excluded a significant role for
other NO·-derived species such as peroxynitrite. Other sources
of H2O2 investigated, such as the aerobic
formation of nitrosoglutathione and the GSH-mediated decay of
nitrosoglutathione, were found quantitatively negligible compared with
the total rate of H2O2 production.
Biological systems of diverse complexity, when exposed to
NO· 1 or stimulated
to produce NO·, usually present changes in the rate of oxygen
free radical production (Ref. 1 and references therein). Decreases in
the rate of oxygen free radical production are often attributed to the
diffusion-controlled reaction between O Considering that under physiological conditions mitochondria constitute
the main intracellular source of oxygen free radicals (2, 3) and that
these organelles can produce NO· (4-8), it becomes of interest
to examine whether the production of mitochondrial ROS is affected by
endogenous NO·. The accurate documentation of the rates of
production of nitrogen and oxygen radicals is an essential step to
understanding the role of these interactions in different, more complex
pathophysiological situations and underlines the relevance of studying
the mechanisms behind these processes in relatively simpler models.
Furthermore, transient changes in ROS production have become an
area of intense research given the growing interest in the role of ROS
as mediators of signal transduction pathways, organ preconditioning
(9), and apoptotic processes (10).
In this study, we examined the rates of O2 free radical
production by intact mitochondria under various conditions of
endogenous NO· production; different mechanisms underlying the
NO·-O Chemicals and Biochemicals
EDTA, EGTA, sodium succinate, sodium malate, sodium glutamate,
mannitol, sucrose, HEPES, bovine serum albumin (fatty acid free), L-arginine, antimycin, 2× crystallized
scopoletin, and desferrioxamine mesylate were purchased from Sigma.
Catalase and horseradish peroxidase (Grade I) were obtained from Roche
Molecular Biochemicals. Oxymyoglobin was obtained as described before
(5, 6). All other reagents were of analytical grade.
Biological Materials
Liver mitochondria were isolated from adult Wistar rats
by differential centrifugation and purified through Percoll
centrifugation (5). This procedure yielded intact mitochondria
minimally contaminated with other subcellular compartments (5).
Essentially, male rats (body weight of 180-220 g) were anesthetized
using a CO2 chamber. The livers were quickly removed;
washed with 0.22 M mannitol, 70 mM sucrose, 0.5 mM EGTA, 2 mM Hepes, and 0.1% defatted bovine serum albumin, pH 7.4 (Buffer A); and homogenized in a 10:1 buffer to
liver w/w ratio. Large cell debris and nuclei were pelleted by
centrifuging at 600 × g for 5 min in a Sorvall SS34
rotor. This supernatant was filtered through two layers of cheesecloth to remove fat. Mitochondria were pelleted by centrifuging the supernatant for 10 min at 10,300 × g in the same
rotor. After suspending the pellet in 5 ml of the previous buffer, the
mitochondrial fraction was loaded on 20 ml of 30% (v/v) Percoll, 0.225 M mannitol, 1 mM EGTA, 25 mM Hepes,
and 0.1% defatted bovine serum albumin, pH 7.4, and spun for 30 min at
95,000 × g in a Beckman 60Ti rotor. Mitochondria,
collected from the lowest band, were washed once with Buffer A and
twice with 0.15 M KCl. Mitochondrial pellets were gently
suspended in a small volume of ice-cold Buffer A. The respiratory
control and phosphate to oxygen ratios of these purified
mitochondria were 6.5 ± 0.2 and 2.8 ± 0.1, respectively, assessed in 0.225 M sucrose, 5 mM
MgCl2, 20 mM KCl, 10 mM potassium phosphate, and 20 mM Hepes/KOH, pH 7.4 (reaction medium)
supplemented with 10 mM succinate and 0.25 mM ADP.
Biochemical Analyses
Oxygen Consumption--
The oxygen uptake of mitochondria was
measured using a Clark-type O2 electrode from Hansatech
(King's Lynn, UK) at 30 °C (5, 7). Intact, purified mitochondria
(0.5-2 mg of protein/ml) were maintained in 1 ml of reaction medium in
the presence of 10 mM succinate as substrate and, where
indicated, different amounts of L-Arg or
NG-monomethyl-L-arginine (NMMA).
NO· Detection--
The production of NO· by
mitochondria (1-2 mg/ml) was evaluated as the change in the absorbance
at 581-592 nm at 22 °C using a dual wavelength, double beam
SLM-Aminco DW-2C UV-visible spectrophotometer. The sample
cuvette contained 2 ml of reaction medium, 0.1-0.3 mM
oxymyoglobin, 10 mM succinate, and 0.1 mM
L-arginine under constant stirring. When NMMA was used in
the incubation, the protein concentration was 3-5 mg/ml. Ten
µM catalase and superoxide dismutase were also present in
the reaction medium to avoid unspecific side reactions (namely, the
reaction of NO· with the superoxide anion or of oxymyoglobin
with peroxynitrite/hydrogen peroxide); however, no significant changes
were found with or without these enzymes. The rates of NO·
production were taken during the first 4-5 min when the ratios of
myoglobin:catalase (in heme):NO· were 28:6:1, thereby avoiding a
significant, if any, inhibition of catalase by NO·. The
L-citrulline production was monitored by a modified
colorimetric technique from Prescott and Jones (11).
Hydrogen Peroxide Production--
The rate of
H2O2 production by intact mitochondria
(0.12-0.15 mg of protein/ml) was measured in reaction medium with 5 µM horseradish peroxidase and 1 mM scopoletin
(12). This high concentration of scopoletin was required to compete
with GSH for the horseradish peroxidase-H2O2
complex (data not shown). The recovery of H2O2 was 80-90% assayed with glucose/glucose oxidase.
Detection of GSNO--
Sample preparation proceeded
as follows. Aliquots of the reactions were withdrawn at
different time points. The aliquots were treated to avoid thiol
oxidation or thiol-disulfide exchange, to terminate any metabolic
process, and to prevent any artifactual GSNO production. To satisfy
these criteria, the samples were kept on ice protected from light
during the duration of the procedure and promptly precipitated with
perchloric acid in the presence of a metal chelator, desferrioxamine
mesylate. The samples (0.8 ml) were treated with 20 µl of 10 mM desferrioxamine mesylate and 36 µl of 70% perchloric
acid, kept on ice for 10 min, and centrifuged at 14,000 rpm for
10 min at 4 °C. The supernatants were treated with 1 volume of 0.15 M K2HPO4, 10 mM sodium
citrate, and 5 mM EDTA (phosphate-citrate-EDTA). The
samples were kept on ice for 30 min and centrifuged at 14,000 rpm for
15 min at 4 °C. The supernatants were filtered through a 2-µm
filter and kept at Protein Determination--
Protein concentration was determined
by biuret assay (15) with the modifications introduced by Yonetani
(16), using bovine serum albumin as the standard.
Statistics--
Data were expressed as mean ± S.E. and
were evaluated by one-way analysis of variance (ANOVA).
Endogenous Nitric Oxide Modulates Hydrogen Peroxide Production by
Mitochondria--
The addition of increasing concentrations of
L-Arg to intact, coupled mitochondria in State 4 (using
either glutamate-malate or succinate) stimulated the production of
NO· (Fig. 1A). The
initial velocity plotted versus L-Arg
concentration represented a right rectangular hyperbola; the
Vmax and Km values were 1.5 nmol of NO· × (min · mg protein)
The rates of NO· production in mitochondria,
maintained in State 4, were decreased significantly when NMMA was
included in the reaction mixture (Fig. 1A). The addition of
this compound did not affect the Vmax but
increased the apparent Km values (15 and 30 µM, respectively). These results indicated that this
compound is acting as a competitive inhibitor of mitochondrial NOS, as has been shown by other reports (17-19). The
KI was calculated from the slope of a plot of
Km(app) versus [NMMA] (Fig. 1B), resulting in a value (15 µM) similar to
that reported for mouse-inducible NOS (13 µM)
(18). Even at high concentrations of NMMA (0.5 mM), a
slow rate of NO· was detectable (0.02 nmol of NO· × (min
· mg protein)
To examine whether endogenous NO· affected the mitochondrial
production of oxygen free radicals, the production of
H2O2 and the oxygen consumption of
intact mitochondria were assessed under different conditions of
NO· generation. Mitochondria were supplemented with either
malate-glutamate or succinate during the experiments to ensure a
nonlimiting pool of NADPH, required for the activity of NOS.
The addition of L-Arg to intact, coupled
substrate-supplemented mitochondria increased the rate of
H2O2 production in a dose-response manner (Fig.
2A). The concentrations of
L-Arg required for the half-maximal effect were 29.6 µM (glutamate-malate) and 34 µM (succinate). At saturating concentrations of L-Arg, the
rate of H2O2 production increased by 42%
(glutamate-malate) and 58% (succinate), whereas the respiratory rates
decreased 33% (glutamate-malate) and 27% (succinate) (Fig.
2B). The concentrations of L-Arg required for
the 50% inhibition of the respiratory rates were 24.7 µM
(glutamate-malate) and 31.5 µM (succinate). The effects
observed on the respiratory rates and H2O2
production were completely reversed by adding 0.2-1 mM
oxymyoglobin to the reaction mixture (not shown). These latter experiments might wrongly indicate that the rate of
H2O2 production was decreased by the reaction
of H2O2 with oxymyoglobin via its pseudocatalatic/peroxidatic activity, without the participation of
NO·. This possibility seemed unlikely based on the following
evidence. First, the rate constants for the reaction of oxymyoglobin or oxyhemoglobin with H2O2 in their peroxidatic
and catalatic activities are 4-5 orders of magnitude smaller than
those corresponding to the reaction of oxymyoglobin with NO·
(21-23), indicating that this molecule will preferentially react with
oxymyoglobin rather than with H2O2. Second, the
addition of NMMA, which blocks the production of NO· by
mitochondrial NOS (independently of the presence of
oxymyoglobin) decreased the rate of H2O2
production, excluding the role of oxymyoglobin in the modulation of
H2O2 production by NO·-producing
mitochondria (see below).
The addition of NMMA to L-Arg- and succinate-supplemented
mitochondria decreased the rate of H2O2
production and increased the respiratory rates in a dose-response
manner (Fig. 3, A and B, respectively). The concentrations of NMMA required for
the half-maximal rate of H2O2 production were
13.9 (glutamate-malate) and 13.8 µM (succinate); at
saturating concentrations of NMMA, the rate of
H2O2 production was decreased 42%
(malate-glutamate) and 37% (succinate). The concentrations of NMMA
required for the half-maximal respiratory rates were 15.7 (glutamate-malate) and 10.2 µM (succinate); at saturating
concentrations of NMMA, the respiratory rates were increased by 85%
(malate-glutamate) and 39% (succinate). The effects observed on the
respiratory rates and H2O2 production mediated
by NMMA were completely reversed by adding increasing amounts of
L-Arg (not shown).
The reciprocal association between ROS and NO· production with
the O2 uptake (Figs. 1-3, Table
I) and the similar concentrations of NMMA and L-Arg required for the half-maximal effects for
the NO· production, respiratory rates, and
H2O2 production (Figs. 1-3) indicated that
these events were biochemically linked, acting at a common site. The
involvement of NO· in these effects was supported by the
reversibility of the effects observed with NMMA (upon the addition of
L-Arg) and L-Arg (upon the addition of
oxymyoglobin). These results also precluded a significant role for
peroxynitrite because this species causes irreversible damage of
respiratory chain components (24). Furthermore, the fact that
oxymyoglobin reversed the modulation of the O2 uptake and
ROS production not only excluded ONOO
Although the association between respiratory rates, hydrogen peroxide
production, and NO· generation was apparent from these results,
no quantitative correlation was demonstrated. Moreover, given the fact
that mitochondria are endowed with a pool of GSNO2
and that its synthesis (25) and decay may produce oxygen
radicals (26) at rates that may be significant, we sought to explore these processes using quantitative strategies.
Role of S-Nitrosoglutathione Metabolism in the Hydrogen Peroxide
Generation by Mitochondria--
The major mitochondrial thiol is
constituted by glutathione (about 10 mM) (27). Glutathione
is synthesized in the cytosol, imported to the mitochondria, and
involved in a protective role against oxidative stress (28). Thiols
react with NO· and/or its metabolites, resulting in the
formation of stable NO· donors or nitrosothiols (29). Recent
reports suggest that the formation of GSNO in vivo
may include the production of O
The decay of GSNO may result in H2O2 production
if the pathway indicated in Fig.4 is
operative. To evaluate the contribution to the rate of
H2O2 by GSNO decay, the rate of
H2O2 generation was evaluated in a control
(mitochondria treated with NMMA for 30 min) and in GSNO-depleted
mitochondria (mitochondria incubated with NMMA at 25°C for 3-4 h
until the concentration of GSNO was not detectable by HPLC). No
significant difference was found in the rates of
H2O2 production between GSNO-depleted and
control mitochondria (98 ± 30 and 137 ± 30 µM/min,
respectively; mean ± S.E.; n=3), indicating that the decay
of GSNO did not contribute significantly to the
H2O2 generation by mitochondria; however, it
did not preclude in vivo situations in which higher
concentrations of GSNO may be achieved, and its decay may have a
substantial contribution to the rate of H2O2
production.
Interaction of Nitric Oxide and Respiratory Chain
Carriers--
We investigated whether the modulation of ROS by
NO·-producing mitochondria was the result of the interaction
between NO· and certain electron carriers of the respiratory
chain. This hypothesis is based on the high affinity of NO· for
metallo-containing proteins (31, 32) and on the fact that certain
inhibitors of the respiratory chain (those that increase the
steady-state reduction level of electron carriers at the NADH dehydrogenase and ubiquinol-cytochrome c reductase segments)
enhance the production of ROS.
Toward this end, the steady-state reduction of the carriers of the
respiratory chain was evaluated in NO·-producing mitochondria by
visible spectroscopy (Fig. 5). Under these conditions, 77% of cytochromes b and c was reduced,
whereas 50% of cytochrome oxidase was reduced. The steady-state
reduction of cytochrome oxidase (in percentage) was found within the
experimental range of the inhibition of the oxygen consumption under
identical conditions. This result agreed with those previously obtained in which changes in the respiratory rates correlated with those in
cytochrome oxidase activity under identical conditions of NO·
production (7).
Experimental evidence for the association between the
increased reduced state of the respiratory chain carriers and
H2O2 production in NO·-producing
mitochondria was provided by evaluating the steady-state reduction of
the respiratory chain carriers and the rate of ROS production under a
variety of conditions, including those that entailed changes in
NO· production (Fig. 6). A
significant correlation was found between the reduction level of
cytochrome b (Fig. 6) or other components of the respiratory
chain (not shown) and the production of H2O2 (Fig. 6) (r = 0.958), indicating that an increased
reduction of these carriers under a variety of conditions (including
those in which NO· is produced) leads to an increased rate of
ROS production.
Based on our results, it seemed that the modulation of ROS
production was accomplished through the reversible inhibition of the
mitochondrial respiratory rate by NO· at the cytochrome oxidase
level. The transient decrease (or inhibition, depending on the
concentration of NO· and oxygen) in the respiratory rate leads
to an increased steady-state reduction of the respiratory chain
carriers, which in turn by reacting with oxygen resulted in the
production of ROS. It is likely that cytochrome oxidase represents the
crossover point in NO·-producing mitochondria (Fig. 5),
excluding a significant interaction of NO· with other available
components of the respiratory chain (cytochromes c or
b) under these experimental conditions. However, if
cytochrome oxidase were the crossover point in this process, it would
have been fully reduced. This apparent discrepancy can be bridged by considering that the interaction of NO· with cytochrome oxidase
is complex, constituted by the reversible binding of NO· to the
heme a3 of cytochrome oxidase and by the reduction of NO· to N2O by this oxidase (7).
Considering the results presented in this study, it could
be wrongly surmised that the effect of NO· on the respiratory
chain is not different from that of other xenobiotics used to enhance
ROS production in mitochondria (e.g. NADH-supplemented
mitochondria plus rotenone or FADH2-supplemented mitochondria plus antimycin). Nitric oxide has two distinctive properties from the aforementioned inhibitors; it is endogenously produced (4-8), and it has a transient effect (5, 7). This latter
property, based on its cytochrome c oxidase-mediated
catabolism, allows a slow but continuous flow of electrons through the
chain, even when a complete suppression of the O2
consumption has been accomplished. This type of modulation of the
respiratory chain has the advantage of avoiding a complete reduction of
the components of the respiratory chain and the subsequent burst of ROS
when lower ratios of [NO·]/[O2] are encountered.
Conclusions--
The mitochondrial production of ROS has been
considered as a side process of the normal oxidative metabolism, its
rate ranging between two levels determined by the mitochondrial
metabolic states, namely States 4 (maximum) and 3 (minimum) (2). This
study demonstrated that the mitochondrial production of ROS is not
limited to these two values and may exhibit a degree of values
modulated by endogenous NO·. Cellular conditions that would
affect the availability of either L-Arg or other cofactors
required for NOS activity would generate variable amounts of NO·
and O
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C until HPLC analysis, usually
performed during the same day. The samples were analyzed by HPLC using
a modified method from Stamler and Feelisch (13). The separation and
detection of GSNO were performed by using a C18 Aqua HPLC column
(column specifications: length and inner diameter, 250 mm × 4.6;
particle size, 5 µm) (PhenomenexTM, Torrance, CA) connected
with a SecurityGuardTM column from the same manufacturer (4 × 3 mm C18 cartridge). The different components were eluted with an
isocratic gradient of 99% 0.1 M monochloroacetic acid,
0.125 mM EDTA, 1.5 mM sodium octyl sulfate, pH
2.8, and 1% acetonitrile. The mobile phase was purged with
helium, and the flow rate was set at 0.8 ml/min. The chromatography equipment consisted of a Class-VP Shimadzu HPLC system
equipped with two high pressure LC10ADvp pumps and an
automatic injector Sil-10ADvp. The chromatograms were
analyzed using the software provided with the system (version 5.03).
The peaks were monitored by a photodiode array detector (215 and 336 nm) and an electrochemical detector (L-ECD-6A from Shimadzu set at
300 mV) connected in series. The identification of the derivatives was performed by comparison with synthetic GSNO. This latter compound was prepared according to Hart (14) by acid-catalyzed nitrosation of
GSH (Elemental analysis: Found, C, 34.5; H, 4.9; N, 15.2; S, 9.0;
H2O, 0.94;
C10H16N4O7S·
H2O requires C, 34; H, 5.1; N, 15.8; S, 9.1;
H2O, 5.1%). Fresh solutions of GSNO were
prepared immediately before each experiment, and the concentrations
were confirmed spectrophotometrically at 334 nm.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 and 5 µM, respectively. Comparable results were obtained by
measuring L-citrulline, cosynthesized with NO· by
NOS (Vmax = 1.2 ± 0.2 nmol of
L-citrulline/min × mg of protein and
Km = 8 µM). The rate of NO·
production was negligible at L-Arg concentrations of
50-100 × Km. This effect was accompanied by
an increase in the rate of oxygen consumption (3-5 times), found to be
resistant to ADP stimulation, and comparable with the increase obtained in the presence of carbonyl cyanide
p-trifluoromethoxyphenylhydrazone. These experimental
observations indicated that the lack of NO· production at high
concentrations of L-Arg was attributed to the uncoupling of
mitochondria by this amino acid.
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Fig. 1.
Nitric oxide production by
L-arginine-supplemented mitochondria. The rate of
NO· production was followed by the oxidation of oxymyoglobin or
by the production of L-citrulline (not shown), as described
under "Experimental Procedures." A, the velocity of
NO· production versus L-Arg concentration
with no (closed circles), 30 µM (closed
triangles), and 75 µM (open circles)
NMMA. B, from the slopes of reciprocal plots of velocity
versus [L-Arg], the apparent
Km was calculated at each concentration of NMMA.
These values were plotted versus the [NMMA].
1) not accompanied by
L-citrulline production. This slow rate has been attributed
to the nonenzymatic release of NO· from GSNO in the presence of
reduced glutathione and superoxide dismutase.2
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Fig. 2.
Effect of L-arginine on
O2 uptake and H2O2
production by intact mitochondria. The
H2O2 production (A) and
O2 uptake (B) of intact, purified mitochondria
were measured in the presence of 5 mM glutamate/0.5
mM malate (closed circles) or 10 mM
succinate (open circles). The O2 uptake was
measured using 1 mg of protein/ml suspended in reaction medium as
described under "Experimental Procedures." The rate of
H2O2 production was measured using 0.12 mg of
protein/ml in reaction medium using the horseradish
peroxidase-scopoletin method. Other experimental conditions were
described under "Experimental Procedures."
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Fig. 3.
Effect of NMMA on O2 uptake and
H2O2 production by intact mitochondria.
The H2O2 production (A) and
O2 uptake (B) of intact, purified mitochondria
supplemented with 0.1 mM L-Arg were measured in
the presence of 5 mM glutamate/0.5 mM malate
(closed circles) or 10 mM succinate (open
circles). Other experimental conditions were described in the
legend to Fig. 1 and under "Experimental Procedures."
but also the
involvement of other NO·-derived species, such as nitrate, also
produced in the presence of oxymyoglobin (23).
Oxygen consumption, H2O2 production, and NO·
generation in intact mitochondria
(Eq. 1)
If this mechanism had been active in mitochondria, assuming oxygen
is the only suitable electron acceptor for Equation 2, increases in
NO· production would have been followed by increases in the
rates of O
(Eq. 2)
1. According to the mechanism outlined
before, if this rate were equivalent to that of O
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Fig. 4.
Synthesis and decay of GSNO.
GS·, glutathionyl radical; GS ,
glutathionyl anion; SOD, superoxide dismutase.
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Fig. 5.
Steady-state reduction of components
of the respiratory chain under different conditions. Rat liver
mitochondria were isolated as described under "Experimental
Procedures." The respiratory control ratio and phosphate to
oxygen ratio number of these preparations were 4.7 and 2.1, respectively, assayed in the presence of 10 mM succinate
and 0.45 mM ADP in reaction buffer (0.225 M
sucrose, 10 mM potassium phosphate, 5 mM
MgCl2, 20 mM Hepes, pH 7.4). The absorption
spectra were recorded using an SLM-Aminco DW-2C UV-visible
spectrophotometer as described previously (33). The difference spectra
of samples, which contained 1 mg of mitochondrial protein in reaction
buffer, were recorded under the following conditions: 20 mM
succinate (State 4; striped bar), succinate plus 0.5 mM ADP (State 3; white bar), succinate and 50 nM antimycin (State 4 plus antimycin; gray bar);
and succinate and 0.15 mM L-arginine (State 4 plus L-arginine; black bar). The concentrations
of each carrier were calculated using the following extinction
coefficients: cytochromes c-c1,
550-540 = 19 mM
1
cm
1; cytochromes bK-T,
564-575 = 20 mM
1
cm
1; cytochromes aa3,
605-630 = 16 mM
1
cm
1.
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Fig. 6.
Rate of H2O2
production by mitochondria and the steady-state reduction of cytochrome
b. The rates of H2O2
production and the steady-state reduction of cytochrome
bK-L were evaluated in mitochondria under State 1, State 4 (succinate or malate-glutamate), State 3 (succinate or malate-glutamate
plus ADP), State 3u (succinate or malate-glutamate plus carbonyl
cyanide p-trifluoromethoxyphenylhydrazone), State 4 plus L-Arg, and State 4 plus NMMA. Other experimental
details were described under "Experimental Procedures" and in the
legend to Fig. 5.
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ACKNOWLEDGEMENTS |
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We thank T. Githu and M. Raveendranathan for technical assistance.
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FOOTNOTES |
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* This work was supported by Grant MCB 9724060 from the National Science Foundation and by a grant from the United Mitochondrial Disease Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of the Undergraduate Research Opportunity Program
fellowships from the University of Minnesota.
§ Recipient of the Margaret Mitchell Award from the National Cancer Institute.
¶ Supported by the Swenson Research Summer Program and the Research Corporation, Cottrell College Science Awards (9906909).
To whom correspondence and reprint requests should be
addressed: Dept. of Chemistry, University of Minnesota, 10 University Dr., Duluth, MN 55812. E-mail: cgiulivi@d.umn.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M007625200
2 M. Steffen, T. Sarkela, A. A. Gybina, T. W. Steele, N. J. Traaseth, D. Kuehl, and C. Giulivi, Biochem. J., in press.
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ABBREVIATIONS |
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The abbreviations used are:
NO·, nitric
oxide;
ROS, reactive oxygen species;
O
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REFERENCES |
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