From the
Aconitase is a member of a family of iron-sulfur-containing
(de)hydratases whose activities are modulated in bacteria by superoxide
radical (O)-mediated inactivation and iron-dependent reactivation. The
inactivation-reactivation of aconitase(s) in cultured mammalian cells
was explored since these reactions may impact important and diverse
aconitase functions in the cytoplasm and mitochondria. Conditions which
increase O production including exposure to the redox-cycling agent
phenazine methosulfate (PMS), inhibitors of mitochondrial
ubiquinol-cytochrome c oxidoreductase, or hyperoxia
inactivated aconitase in mammalian cells. Overproduction of
mitochondrial Mn-superoxide dismutase protected aconitase from
inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase
activity was reactivated (t of 12 ± 3 min) upon removal
of PMS. The iron chelator deferoxamine impaired reactivation and
increased net inactivation of aconitase by O. The ability of
ubiquinol-cytochrome c oxidoreductase-generated O to
inactivate aconitase in several cell types correlated with the fraction
of the aconitase activity localized in mitochondria. Extracellular O
generated with xanthine oxidase did not affect aconitase activity nor
did exogenous superoxide dismutase decrease aconitase inactivation by
PMS. The results demonstrate a dynamic and cyclical O-mediated
inactivation and iron-dependent reactivation of the mammalian
[4Fe-4S] aconitases under normal and stress conditions and
provide further evidence for the membrane compartmentalization of O.
Several growth impairments caused by excess superoxide radical
(O) production
(1, 2, 3, 4, 5, 6) or SOD
A balanced mechanism of O-dependent
inactivation and iron-dependent reactivation modulates aconitase
activity and citric acid cycle activity in E.
coli(9, 10) . Similar inactivation-reactivation
mechanisms may control the activity of other
[4Fe-4S]-dependent metabolic
pathways
(5, 8, 11, 12, 19) , and
regulators of transcription
(20) and translation (21-26).
The conservation of in vitro O
sensitivities
(6, 9, 15, 27) ,
reactivatabilities
(9, 10, 28) , and primary
structures
(29, 30, 31) between the mammalian
mitochondrial and cytoplasmic aconitases and the prokaryotic E.
coli aconitase led us to suppose that similar dynamic
inactivation-reactivation of the aconitases may occur in mammalian
cells and, moreover, that these reactions may be important for
aconitase function in the citric acid cycle and in the control of
cellular iron metabolism
(21) under normal and stress
conditions.
We now describe a O-dependent inactivation and
iron-dependent reactivation of the mammalian aconitases and describe
the use of aconitases as measures of O produced in the mitochondria and
cytoplasm of cultured mammalian cells. The possible roles of a dynamic
inactivation-reactivation in aconitase function are discussed.
Thus,
we tested the effects of various combinations of these
ubiquinol-cytochrome c oxidoreductase inhibitors on aconitase
activity in several mammalian cell types. As shown by Fig. 2,
blocking mitochondrial respiration with antimycin A caused modest but
significant (p < 0.05) decreases in aconitase activity in
all cell types tested. The amount of inactivation ranged from
Together the results demonstrate a significant contribution of O to
aconitase inactivation under steady-state conditions and demonstrate
the importance of the balanced iron-dependent reactivation of aconitase
for the maintenance of aconitase activity in cultured mammalian cells.
In comparison,
H
We have demonstrated a dynamic mechanism of O-dependent
inactivation and iron-dependent reactivation of aconitase in mammalian
cells. Aconitase was inactivated by conditions which increase the
production of O (Fig. 1-3) and was protected from
inactivation by MnSOD (Fig. 3B). Inactivated aconitase
was rapidly reactivated, and reactivation was dependent upon available
iron as evidenced by the inhibition of reactivation by the iron
chelator deferoxamine (Fig. 5A and 6A).
Moreover, overexpression of MnSOD partially protected against the loss
of aconitase activity in the presence of deferoxamine
(Fig. 6B), thus revealing the constant rate of
O-mediated inactivation of aconitase occurring at physiological O
levels. A
The
effects of O and iron on the cytoplasmic and mitochondrial aconitases
cannot be precisely discriminated from measurements of total cell
aconitase activity. Nevertheless, our results indicate that both
cytoplasmic and mitochondrial aconitases are inactivated by O and
subsequently reactivated in vivo. Thus, PMS inactivated
>90% of the total aconitase activity in A549 cells. Since 62% of the
total activity in A549 cells is located to the cytoplasm, we can
conclude that the cytoplasmic aconitase was inactivated. On the other
hand, the inactivation of aconitase by mitochondrion-specific
generators of O in several cell types ( Fig. 2and
Fig. 3B) and the protection against this inactivation by
mitochondrial MnSOD (Fig. 3B) demonstrates an O
dependent inactivation of the mitochondrial aconitase.
Our results
are consistent with a limited diffusibility of O across membranes
(52) and a compartmentalization of O. Thus, high levels of
extracellular O or extracellular SOD failed to modulate aconitase
activity in the cytoplasm or mitochondria of A549 cells. Furthermore,
the fractional loss of total aconitase by antimycin A/FCCP-elicited
mitochondrial O production ( Fig. 2and 3B) correlated
with the enrichment of aconitase activity in mitochondria, suggesting O
production and compartmentalization within the mitochondria. Correction
for the fraction of aconitase localized to mitochondria
(Fig. 4B) reveals similar losses of mitochondrial
aconitase of 77 ± 8% (average ± S.D.) in the four cell
lines examined.
An aconitase inactivation-reactivation cycle showing
the opposing roles of O and iron in modulating aconitase activity is
depicted in Fig. SI. Active mitochondrial aconitase, and
presumably other homologous aconitases, contain
[4Fe-4S]
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
The cyclical inactivation-reactivation of aconitase(s) may
modulate aconitase function(s) under stress and normal conditions. The
citric acid cycle flux capacity and homeostatic regulation of iron
metabolism may be affected during oxidative
stress
(24, 36) , nutritional or immune-induced iron
deficiencies, or SOD deficiencies. In addition, the control of the
aconitase iron-sulfur state by intracellular [O] and
[Fe
Further explorations
of the effects of O on aconitase(s), the citric acid cycle, and
cellular iron metabolism may shed light on the complex interactions of
O and iron in normal and pathological states. In addition, the
aconitase(s) can now be utilized as versatile compartmentalized sensors
for the assay of O.
We thank Dr. Ting-Ting Huang for assistance in the
preparation of the MnSOD overexpressing A375 clones and Dr. Ye-Shih Ho
for kindly providing human MnSOD cDNA. We thank Drs. Irwin Fridovich,
Anne Gardner, and Kamuda Das for careful reading of the manuscript and
for suggestions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
deficiencies
(5, 6, 7) in
Escherichia coli can be attributed to the O-mediated
inactivation of [4Fe-4S]-containing
(de)hydratases
(5, 6, 8, 9, 10, 11, 12, 13, 14) .
Loss of sensitive enzymes including
,
-dihydroxyacid
dehydratase
(8, 11, 12, 15) ,
6-phosphogluconate dehydratase
(5) ,
aconitase
(6, 9, 10) , and fumarases A and
B
(13, 14) collectively impedes branched-chain amino
acid synthesis, the Entner-Doudoroff pathway, and Krebs' citric
acid cycle and may also cause pleiotropic
effects
(16, 17) . Knowledge of these and other sensitive
targets
(18) continues to illuminate mechanisms of toxicity and
the adaptive defense against O.
Cells and Reagents
The human epithelial lung
cell carcinoma A549 (CCL 185), the murine fibrosarcoma L929 (CCL 1),
and human A375 melanoma cells (CRL 1619) were obtained from the
American Type Culture Collection (Rockville, MD). Rat 1A fibroblasts
were provided by G. Johnson of this department. MnSOD overexpressing
A375 clones, D5 and D11, were constructed by transfecting A375 cells
with the expression vector pcDNAI/neo (Invitrogen, San Diego)
containing the 1.0-kilobase EcoRI human MnSOD cDNA fragment
from pSP65-HMS
(32) ligated into the EcoRV site in the
sense orientation; and the control clone, neo5, was created by
transfecting cells with pcDNAI/neo.(
)
Stable
transfectants were selected in medium containing 450 µg/ml
Geneticin, and independently-derived clones were screened for elevated
MnSOD by measuring activity in cell lysates
(34) .
PMS, antimycin A, FCCP, barium (±)-fluorocitrate, fetal
calf serum, bovine milk xanthine oxidase, horse heart cytochrome
c
, nitro blue tetrazolium, riboflavin, and
hydrogen peroxide were from Sigma. Beef liver catalase (260,000
units/ml) and myxothiazole were obtained from Boehringer
Mannheim.Bovine erythrocyte Cu,ZnSOD (3500 units/mg) was from DDI
Pharmaceuticals, Inc., Mountain View, CA. Deferoxamine mesylate was
from CIBA, Summit, NJ. F12K and RPMI 1640 growth media, Hank's
balanced salt solution, trypsin-EDTA, gentamycin sulfate, Geneticin,
and penicillin-streptomycin were obtained from Life Technologies, Inc.
Acetylated cytochrome c was prepared as described
(35) .
Cell Growth, Harvest, and Extract Preparation
Cell
cultures were grown at 37 °C under a humidified atmosphere
containing 5% CO in 10-cm Falcon dishes and cells were
routinely counted with a hemacytometer. A549, Rat 1A, and L929 cell
cultures were grown in F12K medium containing 10% fetal calf serum,
with 100 units/ml penicillin and 100 µg/ml streptomycin. Exposures
to normobaric hyperoxia (pO
of 600 mm Hg) were as described
previously (36). A375 cell clones, neo5, D5, and D11 were grown in RPMI
1640 medium containing 10% fetal calf serum, 10 µg/ml gentamycin
sulfate, and 225 µg/ml Geneticin, and A375 parental cells were
grown in the absence of Geneticin. Cells were harvested and extracts
were prepared as described previously
(36) .
Enzyme and Protein Assays
Aconitase activity was
measured as described previously
(36) using a Beckman DU-64
spectrophotometer equipped with a thermostatted and oscillating
multicuvette holder. SOD activity was assayed in a 0.5-ml assay mixture
containing 10 µM acetylated-cytochrome
c, 50 µM xanthine, 1
mM EDTA, and 200 units/ml catalase in 50 mM potassium
phosphate buffer, pH 7.8, at 25 °C. Xanthine oxidase was added to
produce an initial increase in absorbance at 550 nm of 0.0095 ±
0.03/min. SOD units were calculated from a standard curve prepared with
bovine erythrocyte Cu,ZnSOD using McCord-Fridovich units
(37) .
SOD activities were calculated from the inhibition of cytochrome c reduction between 30 and 60%, a range corresponding to
0.02-0.1 units of SOD activity. Cell extracts were dialyzed
against four changes of 100 volumes of 50 mM potassium
phosphate, pH 7.8, and 0.1 mM EDTA at 4 °C. MnSOD activity
was measured in dialyzed extracts in which Cu,ZnSOD was inactivated by
prior incubation with 50 mM diethyl dithiocarbamate for 60 min
at 30 °C
(38) . Protein concentration was measured by the
method of Bradford
(39) using Coomassie Brilliant Blue staining
reagent (Bio-Rad), and bovine serum albumin, fraction V (Calbiochem, La
Jolla, CA), as the standard. Lactate dehydrogenase and glutamate
dehydrogenase activities were assayed at 25 °C by following the
change of absorbance at 340 nm in a 1-ml reaction mixture containing 50
mM potassium phosphate buffer, pH 7.4, plus 0.2 mM
NADH with either 1 mM sodium pyruvate, or 2 mM
-ketoglutarate and 50 mM NH
Cl, added as the
respective substrates.
Respiration Measurements
Adherent cell cultures
were washed with Dulbecco's phosphate-buffered saline (1.1
mM KHPO4, 8.1 mM
Na
HPO
, 138 mM NaCl, 2.7 mM
KCl, 0.5 mM MgCl
, 0.9 mM
CaCl
) and gently trypsinized. Cells were washed with media
and pelleted at 1000
g for 10 min. Oxygen consumption
was measured using a Clark-type oxygen electrode. Assays were performed
at 37 °C in a 2.0-ml volume of the original growth medium
containing 0.6-1.4
10
cells with respiratory
effectors as indicated. Rates of cyanide-resistant respiration
(40) were measured in the presence of 0.25 mM sodium
cyanide. An O
saturation of 167 µM at 37
°C
(41) corrected for Denver atmospheric pressure of 635 mm
Hg was used for calculations of respiration rates.
Subcellular Fractionation
Six to ten 10-cm dishes
of near confluent cultures (0.5-1.0 10
cells/dish) were washed with 5 ml of ice-cold phosphate-buffered
saline, scraped in 5 ml of phosphate-buffered saline, and centrifuged
in 15-ml conical tubes at 1500
g for 60 s. Cells were
pooled and washed in 1.0 ml of ice-cold fractionation buffer containing
0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 0.1
mM EDTA, 2 mM sodium citrate, and 1 mM
sodium succinate and were centrifuged at 1500
g for 60
s. Cells were resuspended in 0.5 ml of fractionation buffer and
homogenized on ice in a Teflon-glass Duall tissue homogenizer (Kontes
Glass Co., Vineland, NJ) by making 6-7 passes at a maximum speed
of 500 rpm at 4 °C. Cell lysates were centrifuged for 5 min at 1000
g to pellet nuclei, plasma membranes, unruptured
cells, and other debris away from the supernatant containing cytoplasm
and mitochondria. Mitochondria were then separated from the cytoplasmic
components by centrifugation at 10,000
g for 10 min.
The mitochondrial pellet was gently overlaid with 0.1 ml of ice-cold
fractionation buffer and centrifuged at 10,000
g for 2
min to remove residual cytoplasmic protein, and the wash was pooled
with the cytoplasmic fraction. The mitochondria were resuspended in 0.1
ml of ice-cold fractionation buffer, and both fractions were sonicated
for 10 s and clarified by centrifugation at 14,000
g for 30 s. Aconitase activity was assayed immediately since delay
or freezing of extracts under these conditions resulted in the loss of
activity. Extracts were stored at -70 °C, and the respective
cytoplasmic and mitochondrial marker enzymes, lactate dehydrogenase and
glutamate dehydrogenase, were assayed to evaluate fractionations. The
amount of lactate dehydrogenase in the mitochondrial fraction, and
glutamate dehydrogenase in the cytoplasmic fraction, was
16% of the
total in each cell type.
Gel Electrophoresis and SOD Activity
Staining
Samples containing 10 µg of protein were loaded in
a well of a Universal 8 (Ciba-Corning Diagnostics, Inc.) thin film
agarose gel pre-equilibrated with running buffer containing 20
mM Tris glycine, pH 8.2, and 1 mM EDTA. Gels were
electrophoresed at 100 V and 2-3 mA for 60 min and were stained
for SOD activity using the method of Beauchamp and
Fridovich
(42) .
Anoxic Reactivation of Aconitase
Cells were
harvested by gentle trypsinization, washed with medium, and resuspended
at 4 10
cells/ml in the original growth medium with
cycloheximide added at 100 µg/ml. Septum sealed vacutainer tubes
(Becton-Dickinson, 47
10 mm) were filled with 2.6 ml of cells,
and the excess volume was allowed to escape through a syringe needle as
the rubber septum was inserted. Cells in vacutainer tubes were
incubated for 60 min in a 37 °C water bath and were manually
agitated to maintain a uniform cell suspension and to achieve anaerobic
conditions. Aerobic incubations were performed in parallel by adding
cells to 10-cm dishes containing 15 ml of the original medium at 37
°C with 100 µg/ml cycloheximide. Vacutainer tubes were
centrifuged at 7000
g in a microcentrifuge for 2 min,
media was aspirated, and the cell pellet was lysed by sonicating for 10
s in 200 µl of cold lysis buffer containing 50 mM Tris-Cl,
pH 7.4, 0.6 mM MnCl
, and 20 µM
fluorocitrate. Cell lysates were immediately frozen in dry-ice ethanol
and stored at -70 °C.
Exposure of Cells to Xanthine Oxidase and
H
A549 cells were grown to
a density of 4-5 O
10
cells per 10-cm dish and
were washed twice with Hank's balanced salt solution at 37
°C. Washed cell monolayers were incubated at 37 °C for 15 min
in 5.0 ml of Hank's balanced salt solution containing 0.5
mM xanthine and 500 units/ml catalase with or without 25
milliunits of xanthine oxidase, or washed cells were treated with the
indicated concentrations of H
O
in Hank's
balanced salt solution. Cycloheximide was present at 100 µg/ml
throughout the exposures. Rates of O production by xanthine oxidase
were estimated by following the initial rate of cytochrome
c
reduction at 550 nm under mock exposure
conditions with 10 µM cytochrome
c
. H
O
concentrations
were measured spectrophotometrically
(43) .
Data Analysis
The Tukey-Kramer (HSD) statistical
analysis method in the program JMP (SAS Institutes Inc.) was used for
the analysis of significance (p < 0.05) between multiple
pairs.
Inactivation of Aconitase by PMS
A variety of
natural and synthetic quinones, viologens, anthracyclines, and
phenazine pigments increase cellular O and HO
production by diaphorase-catalyzed redox-cycling
mechanisms
(40) . We surveyed several redox-cycling agents for
their ability to increase O production in cultured mammalian cells as
measured by their effects on cyanide-resistant respiration (CRR) and on
aconitase activity. Both are reliable indices of O production in E.
coli(9, 40) . PMS was effective in rapidly
increasing CRR and inactivating aconitase and was therefore selected
for further studies. As shown in Fig. 1A, PMS exposure
elicited a concentration-dependent increase in the CRR of cultured
human epithelial A549 lung cells. Increases in CRR occurred within a
few seconds of PMS addition, and rates were linear for at least 10 min.
PMS increased CRR rates at concentrations in human cells similar to
those required in E. coli(40) . Measurements of
aconitase activity in A549 cells after a 120-min exposure to PMS
revealed proportional decreases in aconitase activity with increases in
CRR rates (Fig. 1B), and >90% of the total cellular
aconitase activity was sensitive to PMS-mediated inactivation. Unlike
the rapid increases in CRR rates, inactivation of aconitase was
progressive during the initial 60 min of exposure to 1.0
µM PMS and then subsequently plateaued
(Fig. 1C), suggesting a relatively slow response of
aconitase activity to elevated O levels upon PMS exposure. Similar
concentration and time-dependent effects of PMS on CRR rates and
aconitase activity were observed in human A375 (neo5) melanoma cells.
Figure 1:
Effect of
PMS on cyanide-resistant respiration and aconitase activity in A549
cells. Panel A, A549 cells grown to a density of 4-5
10
cells per 10-cm dish were measured for CRR in
the presence of 0.25 mM sodium cyanide and the indicated
concentrations of PMS. The respiration rate without cyanide was 35.7
± 4.7 nmol/min/10
cells. Panel B, A549
cells grown to a density of 4-5
10
cells/dish
were treated with the indicated concentrations of PMS for 120 min in
the presence of 100 µg/ml cycloheximide; or Panel C, cells
were exposed to 1.0 µM PMS and harvested at intervals, and
were assayed for aconitase activity. Me
SO, the solvent
control for PMS, was added to a concentration of
0.1% (v/v). Data
represent the average ± S.D. of three independent
exposures.
Inactivation of Aconitase by Inhibitors of
Ubiquinol-cytochrome c Oxidoreductase
Respiratory inhibitors
offered a specific tool for eliciting O production in the mitochondria
and for investigating the interactions of O and aconitase in
mitochondria of intact cultured mammalian cells. Elevated O production
can be elicited by treating respiring submitochondrial particles with
the high affinity ubiquinol-cytochrome c oxidoreductase
inhibitor antimycin A. Protonophores, including FCCP, stimulate O
production by antimycin A-supplemented mitochondria by
>13-fold
(44, 45, 46, 47) .
14%
for A549 cells to
38% for L929 cells. Uncoupling mitochondria with
FCCP stimulated respiration by 2.6-, 1.3-, and 3.3-fold in A549, Rat
1A, and L929 cells, respectively. However, FCCP alone did not
significantly decrease aconitase activity in any cell type. The results
are consistent with the observation that FCCP, and increased
respiration, does not increase O production in isolated
mitochondria
(44, 46) . The combination of antimycin A
and FCCP decreased aconitase activity dramatically to 73.6 ±
1.6, 50.5 ± 0.4, and 32.1 ± 2.3% (average ± S.D.;
n = 3) of its control activity in A549, Rat 1A, and
L929 cells, respectively. These decreases were significant (p < 0.05) relative to measurements with antimycin A alone as well
as those of untreated controls. Myxothiazole is a potent inhibitor
which blocks electron transfer at a site upstream of the antimycin A
site in the ubiquinol-cytochrome c oxidoreductase
(48) and which diminishes O production by antimycin
A-supplemented mitochondrial membranes
(49) . As anticipated,
myxothiazole mitigated aconitase inactivation by antimycin A and FCCP
exposure in all cell types (Fig. 2). By itself, myxothiazole
caused a modest loss of aconitase activity in all cells possibly by
affecting the release of O from the NADH dehydrogenase flavoprotein or
from other reduced upstream sources
(44, 50) .
Figure 2:
Effect of ubiquinol-cytochrome c oxidoreductase inhibitors on aconitase activity in mammalian
cells. A549, Rat 1A, and L929 cells were grown to 90% confluence in
F12K medium and were exposed to 4 µM FCCP, 4
µM myxothiazole (Myx), 0.5 µM
antimycin A (Anti A) individually or in various combinations
in the presence of 100 µg/ml cycloheximide. After a 60-min
exposure, cells were harvested, extracts were prepared and assayed for
aconitase activity. The efficacy of inhibitors was verified by
measurements of cell respiration rates as described under
``Materials and Methods.'' Ethanol was present as the solvent
control at a concentration 0.06% (v/v) in all exposures. 100% aconitase
activity equaled 6.4 ± 0.0, 6.0 ± 0.1, and 18.1 ±
0.3 in A549, Rat 1A, and L929 cells, respectively. Data were normalized
to the control values and represent the average ± S.D. of three
independent exposures. Results are representative of two or more
experiments. The asterisks indicate p < 0.05
relative to the control value.
These
results demonstrate the diminution of aconitase activity under
conditions in which O production is elevated. Since approximately
5.5-fold increases in O production have been measured in intact
isolated mitochondria treated with antimycin A
(44, 47) ,
it seems probable that the depression of total aconitase activity by
14-38%, observed upon exposure of cultured cells to antimycin A
(Fig. 2), was caused by increases in mitochondrial O production
of a similar magnitude.
Elevated Mitochondrial MnSOD Protects Aconitase against
Inactivation by PMS or Antimycin A, but Not by Hyperoxia
MnSOD
activity was measured in A375 cell clones stably transfected with
expression vector alone, or expression vector containing the human
MnSOD cDNA. Control clone neo5 contained 0.8 ± 0.1 units of
MnSOD/mg of protein and the independently-derived MnSOD overproducing
clones D5 and D11 contained 12.8 ± 1.0 units of MnSOD/mg and
13.9 ± 1.1 units of MnSOD/mg of protein (average ± S.D.;
n = 3), respectively. Both clones D5 and D11 produced
MnSOD activity approximately 15-17-fold over control levels and
expressed most of this MnSOD activity in their mitochondria as
demonstrated by subcellular fractionation and SOD gel analysis
(Fig. 3A). Clones neo5, D5, and D11 were used to
investigate further the role of O in aconitase inactivation by PMS, and
antimycin A plus FCCP. As shown in Fig. 3B, antimycin A
plus FCCP or PMS exposure decreased aconitase activity in the control
neo5 clone by 54.6 ± 2.8 and 52.2 ± 5.5% (average
± S.D.; n = 3), respectively. In comparison, the
A375 clones D5 and D11 which overproduce mitochondrial MnSOD were
significantly more resistant to aconitase inactivation by antimycin A
plus FCCP or PMS exposure in all cases (Fig. 3B). These
results directly implicate O in the inactivation of mitochondrial
aconitase by PMS or antimycin A, and they suggest that mitochondrial
MnSOD plays an important role in preventing this inactivation under
normal conditions.
Figure 3:
Effect of elevated mitochondrial MnSOD on
aconitase inactivation in A375 cells. Panel A, cell
mitochondria and cytoplasm were fractionated from six 10-cm dishes of
A375 clones neo5, D5, and D11 grown to a density of 8
10
cells per dish. Cytoplasmic (C) and
mitochondrial (M) extracts (10 µg/lane) were separated by
agarose gel electrophoresis and stained for SOD activity. A375 clones
neo5, D5, and D11 were grown to 6-8
10
cells
per 10-cm dish and were treated with 100 µg/ml cycloheximide and no
additions (
), 0.5 µM antimycin A plus 4
µM FCCP (
), or 1.0 µM PMS (
)
for 60 min (Panel B) and were assayed for aconitase activity.
Percent aconitase activity was normalized to the appropriate control
cell aconitase activity. 100% aconitase activity for controls equals to
2.5 ± 0.1, 3.3 ± 0.1, and 3.1 ± 0.1 milliunits/mg
of protein in neo5, D5, and D11, respectively. Data are the average
± S.D. of three independent exposures. Asterisks indicate p < 0.05 when compared to corresponding neo5
values.
We recently reported the loss of >80% of the
aconitase activity in A549 cells and a 73% loss of aconitase in rat
lungs after a 24-h exposure to normobaric hyperoxia
(36) , and we
hypothesized that this was due to the effect of hyperoxia-induced
mitochondrial O. Interestingly, mitochondrial MnSOD did not protect
aconitase against inactivation during a 24-h exposure of the MnSOD
overexpressing A375 clones to hyperoxia (pO = 600 mm
Hg); aconitase was inactivated to 24.5 ± 0.6, 24.2 ± 2.0,
and 29.9 ± 3.3% (average ± S.D., p > 0.05 for
all pairs; n = 3 per condition) of the air control
activity in neo5, D5, and D11 cells, respectively. The failure of MnSOD
to protect suggests an O-independent mechanism for aconitase
inactivation under hyperoxic conditions possibly involving direct
attack by dioxygen.
Membrane Compartmentalization of Aconitase Activity and
SOD Activity
We examined the subcellular distribution of
aconitase activity since cell-type differences in the localization of
aconitase activity to mitochondria may account for cell-type
differences in aconitase inactivation by antimycin A plus FCCP (
Fig. 2
and Fig. 3B). As shown in
Fig. 4A, total aconitase activity varied considerably
from a low of 1.9 ± 0.1 milliunits/mg of protein in A375 (neo5)
cells to a high of 16.5 ± 0.5 milliunits/mg of total cell
extract protein in L929 cells. There was also significant variation in
the mitochondrial and cytoplasmic distribution of aconitase activity.
The mitochondrial aconitase specific activity was markedly higher than
the cytoplasmic aconitase activity in L929 cells
(Fig. 4A) in which 82.5 ± 0.7% (average ±
S.D.; n = 2) of the total aconitase activity
fractionated with the mitochondria (Fig. 4B). A549 cells
did not show an enrichment of aconitase activity within mitochondria.
Only 37.6 ± 4.7% (average ± S.D.; n = 3)
of the total aconitase activity fractionated with the mitochondria in
A549 cells. A375 (neo5) cells and Rat 1A cells more closely resembled
L929 cells than A549 cells in that their aconitase activity was
enriched in the mitochondrial fraction (Fig. 4, A and
B). These results may explain the greater inactivation of
total aconitase activity in L929, Rat 1A, and A375 (neo5) cells than in
A549 cells since O produced inside the mitochondrial matrix may not
have access to the 62% cytoplasmic fraction of aconitase activity
in A549 cells.
Figure 4:
Cytoplasmic and mitochondrial aconitase
activities in A549, A375 (neo5), Rat 1A, and L929 cells. Aconitase
activity and protein were measured in cytoplasmic, mitochondrial, and
whole cell extracts (Panel A), and the percent mitochondrial
aconitase activity was calculated from the total of mitochondrial plus
cytoplasmic aconitase activities for each cell type (Panel B).
The data in Panel A show the average ± S.D. and are
representative data of two or three determinations for each cell line.
The data in Panel B represent the average ± S.D. of two
or three independent subcellular fractionations for each cell line. The
asterisk indicates p < 0.05 relative to other cell
lines.
We also measured the total SOD and mitochondrial
MnSOD activity in cells since differences in aconitase inactivation may
be, at least in part, due to differences in the amount of SOD. While
total SOD activities were remarkably similar at 2.8 ± 0.2
units/mg (average ± S.D.), the MnSOD activites varied 1.6
± 0.2 (A549), 0.8 ± 0.1 (A375 clone neo5), 0.4 ±
0.1 (Rat 1A), and 0.9 ± 0.1 (L929) units/mg (average ±
S.D.; n = 3). It is interesting to note that while no
gross differences in total SOD activity were detected in these
different cell types, A549 cells contained 2-4-fold more
MnSOD activity per total cell protein. Thus, higher mitochondrial MnSOD
activity in A549 cells may contribute to the greater resistance of A549
cells to aconitase inactivation during exposure to antimycin A plus
FCCP.
Dynamic Iron-dependent Reactivation of Aconitase in
Vivo
To determine whether inactivated aconitase was reactivated
upon removal of the O insult, PMS-treated A549 cell monolayers were
washed and aconitase activity was measured at intervals in the presence
of cycloheximide. Cells recovered 50% of the lost activity within 12
min, and 95% within 75 min (Fig. 5A, line
1). The half-time for reactivation of aconitase (12 ± 3
min) estimated from line 1 was slower than that determined for the
E. coli aconitase (3-5 min)
(9, 10) .
Reactivation in E. coli was shown to be dependent upon iron.
The importance of available iron for mammalian aconitase reactivation
is demonstrated by the effects of the avid iron chelator deferoxamine
on aconitase inactivation, reactivation rates, and total recovery.
Simultaneous exposure of cells to PMS and deferoxamine for 30 min
(Fig. 5A, line 2) resulted in increased
aconitase inactivation, and subsequent incubation of cells with
deferoxamine slowed the reactivation rate and decreased the maximum
aconitase activity recovery. Preincubation of cells with deferoxamine
(60 min), to allow its cellular uptake, caused an even more profound
impairment of aconitase reactivation (Fig. 5A, line
3). Exposure to deferoxamine under steady-state conditions also
produced a slow but progressive loss of cellular aconitase activity
relative to that seen in cells exposed to cycloheximide alone
(Fig. 5B, compare lines 1 and 2).
Deferoxamine also enhanced O-mediated aconitase inactivation during a
2-h exposure of A375 clone neo5 to antimycin A
(Fig. 6A), and A375 clones D5 and D11 producing elevated
MnSOD were more resistant to deferoxamine-enhanced inactivation.
Elevated MnSOD provided a significant 15-17% protection of
aconitase activity during a prolonged 5.5-h incubation with
deferoxamine (Fig. 6B). This experiment reveals the
constant inactivation of aconitase by endogenously produced O under
otherwise normal growth conditions. Iron-dependent reactivation thus
appears to be a primary mechanism to maintain aconitase activity since
even prolonged inhibition of protein synthesis with cycloheximide was
comparatively without effect on aconitase activity in A549 and A375
cells (Fig. 5B and Fig. 6B).
Figure 5:
Effect
of deferoxamine on aconitase reactivation in A549 cells. Panel
A, A549 cells (4-5 10
cells/dish) were
exposed to 1.0 µM PMS for 30 min. Cells were washed twice
with 10 ml of F12K medium (37 °C) and incubated in 10 ml of fresh
medium at 37 °C, and the recovery of aconitase activity was
measured at intervals in cells exposed and maintained either in the
absence (line 1) or presence of 200 µM
deferoxamine (line 2) or in cells treated with 200
µM deferoxamine for 60 min prior to PMS exposure and
maintained with deferoxamine throughout the incubation (line
3). Cycloheximide (100 µg/ml) was present during all exposures
and incubations. Panel B, A549 cells were exposed to
cycloheximide alone (line 1) or with 200 µM
deferoxamine (line 2), and aconitase activity was measured at
intervals. Percent aconitase activity values are normalized to the
control value at the time of PMS exposure. Data are the average
± S.D. of three independent
exposures.
Figure 6:
Effect of deferoxamine on O-mediated
aconitase inactivation in A375 cells. A375 clonal cell lines neo5, D5,
and D11 were grown in RPMI 1640 medium to 6-8 10
cells/dish and treated with 100 µg/ml cycloheximide.
A, cells were incubated alone (
), with 0.5
µM antimycin A (
), or with antimycin A plus 200
µM deferoxamine (
) for 120 min. B, cells
were incubated with (
) or without (
) 200 µM
deferoxamine for 5.5 h and assayed for aconitase activity. Data
represent the mean ± S.D. of three independent exposures.
Aconitase activity is normalized to 100% and equals 2.8 ± 0.0,
3.5 ± 0.1, and 3.6 ± 0.1, and 2.5 ± 0.1, 2.7
± 0.1, and 2.8 ± 0.1 milliunits/mg of protein (average
± S.D.; n = 3) for neo5, D5, and D11, in
panels A and B, respectively. Asterisks indicate p < 0.05 relative to corresponding neo5
value.
A
10-15% inactive, O-sensitive fraction of aconitase was measured
in E. coli during aerobic growth
(9, 10) . This
inactive, but activable, fraction of aconitase presumably reflects the
steady-state balance of inactivation-reactivation. An inactive fraction
of aconitase of 14% was also measured in A549 cells under normal
aerobic incubation conditions as shown by the increase in the specific
activity of aconitase from 5.40 ± 0.04 to 6.30 ± 0.09
milliunits/mg (average ± S.D., p < 0.05; n = 3) in cells incubated anaerobically in the presence of
cycloheximide as described under ``Materials and Methods.''
Effects of Extracellular O and
H
The O-sensitive aconitase provided, for the first
time, a sensitive method for measuring how much O passes through the
membrane of intact cells and for assessing the effect of extracellular
[O] on intracellular [O]. O produced at a
relatively high rate (2 nmol minO
on Intracellular Aconitase
Activity
ml
for 15 min) with xanthine oxidase and xanthine did not
significantly affect total aconitase activity in A549 cells. After
exposure to xanthine oxidase-generated O, treated and control cells
contained 4.84 ± 0.23 and 5.18 ± 0.12 milliunits/mg
(n = 3; ± S.D., p > 0.05) aconitase
activity, respectively. For comparison, this amount of xanthine
oxidase-generated O would be roughly equivalent to that released by 5
10
activated neutrophils applying a rate of 2 nmol
of O min
per 10
neutrophils
(51) .
Moreover, Cu,ZnSOD (200 units/ml) added extracellularly did not protect
aconitase activity during a 60-min exposure to 1.0 µM PMS.
PMS inactivated aconitase to 31.3 ± 0.3% in the absence and to
29.4 ± 0.7% (average ± S.D., p > 0.05; n = 3) in the presence of added Cu,ZnSOD. The results suggest
that the rate of passage of O into or out of the plasma membrane does
not significantly affect intracellular or intramitochondrial
[O] in intact cells.
O
, which freely permeates biological
membranes, did inactivate aconitase in A549 cells during a 15-min
exposure. H
O
added in bolus concentrations of
10, 50, and 100 µM caused a concentration-dependent
decrease of aconitase activity to 94.4 ± 1.4, 92.5 ± 1.1,
and 42.5 ± 2.4% (average ± S.D., p < 0.05 for
all pairs; n = 3) of the control activity,
respectively. However, scavenging H
O
with
exogenously added catalase (1000 units/ml) did not affect the
inactivation of aconitase during exposure of the cells to PMS (1.0
µM; 60 min), thus H
O
appears to
have a minimal role in PMS-mediated aconitase inactivation. Aconitase
activity in catalase-treated and control cells was 28.5 ± 0.4
and 31.3 ± 0.3% (average ± S.D., p > 0.05;
n = 3), respectively.
14% inactive fraction of aconitase activity was detected
in growing A549 cells and presumably reflects the steady-state balance
of inactivation-reactivation under normal growth conditions. Our
results do not rule out the participation of other potentially
important oxidants such as H
O
or O
in aconitase inactivation. However, the requirement of
comparatively high exposure levels in vivo and relatively poor
reactivities in vitro(6, 15) suggest a lesser
role of O
and H
O
in the
inactivation of aconitase under normal growth conditions.
clusters which are attacked and
oxidized by endogenous O with estimated second order rates of 8
10
M
s
to 3
10
M
s
(15, 27) to form inactive aconitases containing stable oxidized
[3Fe-4S]
clusters. Release of the solvent
exposed iron atom in the ferrous state, cluster oxidation, and
formation of H
O
are thought to occur during the
inactivation process
(15) . Regeneration of the active aconitase
[4Fe-4S]
cluster is efficiently achieved
in vivo with a pseudo-first order rate of
0.0014
s
(t =
12 min)
(Fig. 5A) presumably by the univalent reduction of the
oxidized [3Fe-4S]
cluster to form a
[3Fe-4S]
cluster
(28, 53, 54) and subsequent insertion of a ferrous ion from a
deferoxamine-sensitive iron pool.
Figure SI:
Scheme I
A balanced steady-state aconitase
inactivation-reactivation cycle (Fig. SI) can be described by
Equation 1 (see below) where the rate of aconitase inactivation equals
the rate of aconitase reactivation and when the availability of iron
and other reactants is assumed to remain constant. We can thus ideally
solve for [O] from the inactive and active fraction of
aconitase, and the rate constants for inactivation
(k) and reactivation
(K
=
[Fe
] k
) as
shown by Equation 2. Applying the in vitro estimates of
k
(15, 27) and a value
of 0.0014 s
for K
,
we calculate [O] = 50-200 pM when
aconitase is 50% active, and [O] = 8-30
pM under normal conditions when aconitase is 14% inactive and
86% active. These rough approximations of [O] are in the
range of the [O] of
8 pM estimated from rates
of O production and dismutation in mitochondria (55). We can also see
from this relation that the aconitase reactivation rate must be
6
times greater than the rate of aconitase inactivation to maintain 86%
active aconitase and, moreover, that the half-time for aconitase
activity turnover due to oxidative inactivation at a normal balance of
86% active aconitase equals
6
12 or
72 min. Thus, the
steady-state inactivation-reactivation cycle of aconitase turns over
relatively rapidly (t =
72 min) when compared with
aconitase turnover due to aconitase degradation and synthesis
(Fig. 5B and 6B)
(56) . Without absolute
rate constants to apply in vivo, relative changes in
steady-state [O] can be estimated from the change in the
ratio of inactive and active aconitase under X and Y conditions as described by Equation 3. For example, by correcting
for the fraction of aconitase inactivated in mitochondria of cells
exposed to antimycin A/FCCP, and by applying an estimated 14% inactive
fraction of mitochondrial aconitase during normal conditions, we
calculate 20-40-fold increases in steady-state mitochondrial
[O]. Furthermore, we can estimate that the 15-17-fold
elevation of mitochondrial MnSOD in the MnSOD overexpressing A375
clones decreased mitochondrial aconitase inactivation by
60%
(Fig. 3) and the corresponding mitochondrial [O] by
12-fold. It is expected that decreases in available
[Fe
] will alter the balance of
inactivation-reactivation (Equation 1) by decreasing the rate of
reactivation but not the rate of inactivation. Indeed, this effect can
explain the greater loss of aconitase activity in cells treated with
deferoxamine and the protection against this loss by elevated MnSOD
(Fig. 6).
] may also facilitate iron-sensing by
the RNA-binding cytoplasmic aconitase during
homeostasis
(21, 22, 25, 26) .
Interestingly, the opposing effects of iron and O on aconitase, and
other [4Fe-4S]-containing (de)hydratases, can explain the
previously puzzling and paradoxical benefit of MnSOD up-regulation
during iron limitation in E. coli(9) . It may also
provide a rationale for the increase in mitochondrial MnSOD synthesis
(57) which accompanies increased ferritin synthesis
(58) and hypoferremia
(33) during the inflammatory
cytokine-mediated immune response in mammals.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.