Oligomycin sensitivity of mitochondrial
F1F0-ATPase
in diabetes-prone BHE/Cdb rats
Sook-Bae
Kim and
Carolyn D.
Berdanier
Department of Foods and Nutrition, University of Georgia, Athens,
Georgia 30602-3622
 |
ABSTRACT |
BHE/Cdb and
Sprague-Dawley rats differ in their mitochondrial DNA sequence for the
ATPase 6 ("subunit a") gene. Base substitutions in
this sequence result in the substitution of asparagine for aspartate at
position
101 and the substitution of serine for
leucine at position
129. Differences in sensitivity to
oligomycin were observed. When the isolated
F1F0-ATPase
complex was studied and ATPase activity was assessed, that which was
isolated from the BHE/Cdb rats was less sensitive to oligomycin
inhibition than that which was isolated from the Sprague-Dawley rats.
In contrast, when oxygen consumption was measured [oxygen
phosphorylation (OXPHOS)] and a dose-response curve was generated
with isolated mitochondria from these two strains, there was a shift to
the left for the BHE/Cdb rat mitochondria. These mitochondria were more
sensitive to oligomycin inhibition of OXPHOS than were mitochondria
isolated from Sprague-Dawley rats. The OXPHOS results are consistent
with those from human fibroblasts having either a normal or mutated ATPase 6 gene.
oxidative phosphorylation; mitochondria; diabetes
 |
INTRODUCTION |
THE BHE/CDB RAT mimics the human with mitochondrial
diabetes (1-3). It develops moderate hyperglycemia and impaired
glucose tolerance as it ages, and its mitochondrial function
deteriorates (1-3, 5, 6). These traits are maternally inherited
(20). Detailed studies of these diabetes-prone rats have shown that these rats have less efficient coupling of mitochondrial respiration to
ATP synthesis than normal rats (5, 6, 11, 28). Although thyroxine can
increase mitochondrial ATP synthesis in normal rats, via an increase in
the synthesis and activity of proteins in the various components of
oxidative phosphorylation (OXPHOS) (25, 26), it was without effect in
BHE/Cdb rats (4). These results suggested that although thyroxine
increased mitochondrial protein synthesis, the proteins that were
synthesized did not contribute to an increase in ATP production. In
fact, with some substrates, the mitochondria appeared uncoupled. This
observation could be explained by the presence of a genetic error in
the mitochondrially encoded subunits of the
F0-ATPase. Recent studies of the
BHE/Cdb rat have shown that base substitutions at
positions
8204 and
8289 in the mitochondrial ATPase 6 gene exist in this rat (19; C. Herrnstad, unpublished
observations). The inferred amino acid substitutions at
residues
101 and
129 of
F0-ATPase "subunit a," which
is encoded by the ATPase 6 gene, could have an effect on the functional
characteristics of
F1F0-ATPase.
Subunit a provides part of the proton channel. The other part is
provided by subunit 9 (subunit c), a nuclear-encoded subunit. The
inferred amino acid sequences of normal and mutated subunit a suggested
that these amino acid substitutions could affect the functional
characteristics of the
F1F0-ATPase.
This would be consistent with early reports of mutated
Escherichia coli
F1F0-ATPase
(7, 13, 23). We have already reported that hepatic mitochondria from
BHE/Cdb rats were more responsive to the suppression of OXPHOS by the
calcium ion than mitochondria from Sprague-Dawley rats (17). In the
present work, we report on the strain differences in oligomycin
sensitivity of
F1F0-ATPase and of the isolated mitochondria. We hypothesized that the amino acid
substitutions in the subunit a would increase the sensitivity of
mitochondria to oligomycin. Oligomycin blocks proton conductance primarily through its binding to the oligomycin-conferring protein found in the stalk of the
F1F0-ATPase
complex. We hypothesized that oligomycin binding would also involve the
F0 portion of ATPase. Hence, we
examined both the activity of
F1F0-ATPase
complex and OXPHOS with respect to the response to oligomycin. Whereas
the isolated
F1F0-ATPase
from the BHE/Cdb rats was less sensitive to oligomycin inhibition
than that from the Sprague-Dawley rats, when isolated mitochondria were
studied, the reverse was found.
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MATERIALS AND METHODS |
Animals and diets. Two strains of rats
were used: BHE/Cdb (UGA colony) and Sprague-Dawley rats (Harlan
Sprague Dawley, Indianapolis, IN). In each study, the rats were
fed a standard stock diet (laboratory animal chow, Ralston Purina, St.
Lewis, MN) and killed at 150 days of age. They were cared for in
accordance with the guidelines of the National Research Council
as described in the National Institutes of Health Publication
88-23 Guide for the Care and Use of Laboratory
Animals.
Purification of
F1F0-ATPase.
Liver mitochondria, mitoplast, and submitochondrial particles (SMP)
were isolated by the method of Pedersen and co-workers (9, 29).
F1F0-ATPase
was purified by anion-exchange HPLC (Mono Q-HR 10/10 column, Pharmacia)
with the methods developed by Yoshihara et al. (30). Freshly isolated mitochondria (100 mg/ml) in isolation medium (H-medium that contained 220 mM D-mannitol, 70 mM
sucrose, 2 mM HEPES, and 0.5 mg/ml defatted BSA) were treated with 4 mg/ml digitonin on ice for 20 min with stirring. This
mixture was then diluted 3:1 with H-medium and centrifuged at 10,000 g (10 min, 0-4°C). The
resulting sediment was resuspended in one-half of the original volume
in H-medium and centrifuged as described previously. These mitoplasts
were resuspended in H-medium at 50 mg/ml, diluted 1:25 (vol/vol) in cold deionized water to 2 mg/ml, and centrifuged at 10,000 g for 15 min. The sediment was
resuspended to 50 mg/ml in cold, deionized water and sonicated with the
large probe at 95% maximal intensity for 2 min total time in 15-s
intervals at 0-4°C. Large mitochondrial fragments were removed
by centrifugation at 10,000 g. The SMP were sedimented from the resulting supernatant at 100,000 g for 30 min at 0-4°C. The
sediment was resuspended to a final concentration of 50 mg/ml with
H-medium. SMP were added at a concentration of 5 mg of protein/ml with
gentle stirring to PEG buffer [20 mM potassium phosphate, 5 mM
EDTA, and 20% (vol/vol) glycerol (pH 7.4)] at 0°C. Then 1%
n-heptyl
-thioglucoside was added,
and the mixture was stirred gently for 5 min. The
suspension was diluted with an equal volume of PEG buffer and
centrifuged at 105,000 g for 30 min.
The resulting supernatant was concentrated in a Centricon 10 (Centricon
Microconcentrator, Amicon), and then the solvent was changed by adding
an equal volume of PEG buffer and concentrating the solution to its
original volume in the Centricon cell. The procedure was repeated two
times. All procedures were carried out at 0°C. The solution (10 mg
of protein) was applied to MonoQ-HR 10/10 column (Pharmacia),
anion-exchange HPLC, which was equilibrated with PEG buffer. Elution
was carried out with a linear gradient of 0-100% 250 mM potassium
phosphate, 5 mM EDTA, 1 M KCl, and 20% glycerol at pH 7.4 for 60 min.
Fractions containing
F1F0-ATPase were collected and centrifuged at 155,000 g for 2 h at 20°C, and the
precipitate obtained was suspended in a minimum volume of PEG buffer.
Protein determination. Mitochondria
and mitoplast protein were measured by the biuret method. SMP and
purified
F1F0-ATPase protein were measured by the Bradford method. BSA was used as standard
in both cases.
Determination of ATPase activity.
ATPase was assayed by a modification of the method of Pullman and
Monroy (24). The basic incubation mixture (1 ml) consisted of 50 mM
Tris · HCl (pH 8.0), 3.3 mM
MgCl2, 2 mg antimycin A, 1 mM ATP,
and 0.3 mM NADH, 1 mM phosphoenolpyruvate, 5 U of lactate
dehydrogenase, and 2.5 U of pyruvate kinase. The reaction was initiated
by the addition of 10-50 µl of the sample to be
measured. Oxidation of NADH was followed spectrophotometrically at 340 nm at a constant temperature of 30°C. The sensitivity of the ATPase
to oligomycin was measured by the addition of 10 µl of 650 µg/ml
oligomycin solution. Enzyme activity was expressed in terms of
micromoles of ATP hydrolyzed per minute per milligram of protein, which
is equal to the micromoles of NADH oxidized per minute per milligram of
protein. Oligomycin sensitivity was expressed as the percent reduction
in ATPase activity with the addition of oligomycin. To determine the
ability of a monoclonal antibody to ATPase to inhibit the enzyme
activity, 100 µg of monoclonal antibody in PBS were incubated with 10 µg purified
F1F0-ATPase
before the assay of the ATPase activity.
Determination of OXPHOS. Oxygen
consumption was determined with an oxygen electrode (model 5331, Yellow
Springs Instrument, Yellow Springs, OH) with a 2.5-ml chamber and
oxygen meter (UGA Instrument Design Group, Athens, GA). The reaction
chamber was fitted with a magnetic stirrer, and temperature was
controlled at 25°C. The respiration medium consisted of 75 mM
glycine, 10 mM phosphate buffer (pH 7.4), 75 mM KCl, and 10 mM
Tris · HCl (pH 7.2). The medium was equilibrated with
air at 25°C. After the medium was placed in the incubation chamber,
subsequent additions were made with Hamilton syringes through the
capillary aperture on the top of the apparatus. The entry port was kept
sealed at all other times. The following reagents were stored frozen
until needed: 25 mM ADP (pH 6.8), 0.65 M succinate (pH 7.2), and
oligomycin (650 µg/ml). In a typical run, freshly isolated
mitochondria (2.5 mg protein) were added to the incubation medium
containing 5 mM succinate. After ~2 min, 375 nmol of ADP were added
to stimulate respiration. This was repeated at least twice. Then,
graded amounts (0.02-0.1 µg/mg mitochondrial protein) of
oligomycin were added with the ADP, and respiration was again
determined. A separate run for each level of oligomycin was performed.
State
3 and
state 4 oxygen consumption rates were
calculated according to Chance and Williams (10). Respiratory control
ratios and ADP-to-O ratios were calculated according to Estabrook (12).
Statistics. Where appropriate,
the means were compared with SuperANOVA. Two mean comparisons were made
with the Student's t-test.
P < 0.05 was considered significant.
 |
RESULTS |
The assessment of the strain differences in ATPase activity at
each step of the
F1F0-ATPase
isolation and purification is shown in Table
1. HPLC and electrophoresis were used for
this purification. The elution patterns for the ATPase from the two strains are shown in Fig. 1. The strains
differed in this pattern probably due to small differences in the amino
acids that comprise the various subunits of the complex.
Fraction
36 contained the ATPase in the
Sprague-Dawley rats, whereas fraction
33 contained the enzyme complex in the
BHE/Cdb rats. Between fractions
10 and 20, there were additional peaks in the
extracts. The identity of these peaks is unknown. With respect to the
data presented in Table 1, it should be noted that only one level of
oligomycin was used to assess its effect on ATPase activity. The level
used was 65 µg/ml reaction volume. This level of oligomycin was the highest level of inhibitor used in the studies of OXPHOS by the isolated mitochondria. As the ATPase was purified, its specific activity increased as expected. By the completion of the treatment of
the extract with the Centricon, there was an ~32-fold increase in
ATPase activity in the extract. There was a corresponding decrease in
the amount of protein, and this was expected as well. Regardless of the
level of purification, there was a strain difference in sensitivity of
the enzyme to oligomycin inhibition. The ATPase from the BHE/Cdb rats
was less sensitive to oligomycin inhibition than was the ATPase from
the Sprague-Dawley rats. This strain difference was observed at each
step of the isolation. Electrophoresis of the final extract showed that
the subunits of the ATPase separated in stoichiometric amounts. The
bands that were separated from the two strains were similar. With the
use of antibodies produced to peptides synthesized to replicate the
hydrophilic portion of the ATPase subunit a, a progressive inhibition
of the ATPase was shown for both strains (Fig.
2). The maximal inhibition of ATPase activity by the addition of these antibodies was 20% with 60 min of
incubation.

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Fig. 1.
Purification of hepatic mitochondrial
F1F0-ATPase
in BHE/Cdb and Sprague-Dawley (SD) rats. Elution profile from
anion-exchange chromatography on MonoQ-HR column with corresponding
SDS/PAGE analysis. A: BHE/Cdb ATPase.
B: Sprague-Dawley ATPase.
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Fig. 2.
Effects of monoclonal antibodies on hepatic mitochondrial
F1F0-ATPase
activity in BHE/Cdb and Sprague-Dawley rats. Purified
F1F0-ATPase
(10 µg) was incubated at 0°C with monoclonal antibodies (100 µg) for indicated length of time.
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|
Figs. 3, 4,
5, and 6 show
the effects of graded additions of oligomycin to mitochondria being
assessed for their OXPHOS performance. In contrast to the results of
the study of the effect of oligomycin on ATPase activity, there was a
shift to the left in the response of isolated mitochondria from the
BHE/Cdb rats. At doses from 0 to 0.04 µg/mg protein, BHE/Cdb
mitochondria were more responsive to the inhibitory action of
oligomycin with respect to state
3 respiration than were mitochondria
from Sprague-Dawley rats. At the highest level, this strain difference
reversed itself, although the difference between the strains was not
statistically significant.

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Fig. 3.
State 3 respiration of BHE/Cdb and
Sprague-Dawley rats in various concentrations of oligomycin.
* Significant differences (P < 0.05) between strains; n = 6 for each
point.
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Fig. 4.
State 4 respiration of BHE/Cdb and
Sprague-Dawley rats in various concentrations of oligomycin. There were
no significant strain differences due to oligomycin addition.
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Fig. 5.
Respiratory control (RC) ratio of BHE/Cdb and Sprague-Dawley rats in
various concentrations of oligomycin. * Significant differences
(P < 0.05) between strains.
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Fig. 6.
P/O ratio of BHE/Cdb and Sprague-Dawley rats in various concentrations
of oligomycin. * Significant differences
(P < 0.05) between
strains.
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DISCUSSION |
This work is of interest because it shows a strain difference in
response to oligomycin that was dependent on the assay used as well as
the dose of oligomycin used. When the ATPase assay was used, the
mitochondria and the purified enzyme from the BHE/Cdb rats appeared to
be less sensitive than that from Sprague-Dawley rats. When OXPHOS was
assessed, the reverse was observed. That is, the BHE/Cdb mitochondria
appeared to be more sensitive to oligomycin inhibition than
mitochondria from Sprague-Dawley rats. On closer examination of these
two data sets, however, it is apparent that this contrast in results
might be due to the dose of drug used rather than any difference in
response to oligomycin by the isolated ATPase and the OXPHOS
measurements. At the highest dose of oligomycin, OXPHOS was inhibited
in mitochondria from both strains. Because OXPHOS assessment measures
more than just the activity of the
F1F0-ATPase,
the lack of a significant strain difference at the highest oligomycin
dose (the dose used when ATPase activity was determined) could be
understood. In addition, the mitochondrial preparations might have been
contaminated with other ATPase-containing organelles. These would
contribute ADP to the media and contribute an error term to the OXPHOS
measurement. However, electron microscopy of the mitochondrial
preparations showed that such contamination was very small (<1%).
Mitochondrial preparations from both strains were similarly affected.
At the lower levels of oligomycin (0-0.04 µg/mg mitochondrial
protein), the strain differences in
state
3 oxygen consumption were observed, and this shift to the left indicates that BHE/Cdb mitochondria were
respiring far slower than Sprague-Dawley mitochondria and that this
rate of respiration was further slowed by increasing levels of
oligomycin. Thus, regardless of the measurements made, there definitely
was a difference due to strain in the responsiveness to oligomycin.
How does this relate to the known genetic defect in the BHE/Cdb
mitochondrial ATPase 6 gene? Our observation of a left shift in
oligomycin sensitivity is consistent with the report of Vazquez-Memiji et al. (27). These workers studied fibroblasts from humans with either
a T8993G or a T8993C point mutation in the ATPase 6 gene. This mutation
is within 30 bp of the mutation we have observed in the BHE/Cdb rats
(19, 20). In the T8993G mutation and in our rat mutation, there is an
inferred change in polarity of one of the amino acid residues that
protrudes out into the proton channel. In the human T8993G mutant, the
base substitution means a substitution of arginine for leucine. In the
rat, the substitution is asparagine for aspartate. In both the rat and
the human, there is impairment in ATP production efficiency and an
increase in the sensitivity to inhibition by oligomycin. The humans
with the T8993C mutation are less affected with respect to the proton
channel in that the substitution is proline for leucine. ATP production is only slightly reduced, but oligomycin sensitivity is greatly increased.
Oligomycin binding is usually thought to be the responsibility of the
oligomycin-conferring protein located in the stalk of the ATPase, but
the present observations as well as those of Vazquez-Memiji et al.
suggest that the subunit a is involved as well. Oligomycin binding to
the subunit a, in turn, appears to be dependent on the amino acids that
comprise this subunit. Despite the fact that the mutation we have
observed is 10 amino acid residues away from that reported for both
sets of human mutants, the proton channel is, nonetheless, changed and
so too is the responsiveness to oligomycin. That other components of
the ATPase complex might be involved in the responsiveness to
oligomycin should not be discounted. Mutations in one or more subunits
of the F1 or the
F0 or the oligomycin-conferring protein could have effects on ATPase activity and oligomycin
sensitivity. Such has been shown in mutant ovary cells and in mutant
yeast and Escherichia
coli cells (7, 13, 14, 18, 21, 23). Mitochondria isolated from these mutants have shown varying reductions in ATPase activity, OXPHOS, and oligomycin sensitivity. In some instances, the mutants have reduced oligomycin sensitivity, and in
other mutants, oligomycin sensitivity is increased. As described previously for the two human mutants and in the two rat strains, varying degrees of oligomycin sensitivity occur in these species as
well. In the BHE/Cdb rat, there may be more mutations in the genes that
encode the ATPase that could affect the mitochondrial characteristics.
We have already reported on their increased sensitivity to the calcium
ion (17) and on strain differences in OXPHOS in animals fed different
carbohydrates and fats (11, 16, 28). Saturated fat in the diet
potentiates the strain difference in OXPHOS (16). Others (8) have also
reported that increases in cholesterol can affect mitochondrial ATPase activity.
Altogether, the present data on differences in oligomycin sensitivity
plus those previously reported provide considerable insight into the
role mitochondria play in the control of metabolism, especially the
metabolism of glucose.
 |
ACKNOWLEDGEMENTS |
This study was supported by Georgia Agricultural Experiment Station
Project no. H947, The Bly Memorial Fund, and The UGA Diabetes Research Fund.
 |
FOOTNOTES |
Present address of S.-B. Kim: 912-402 Mokdong APT,
Shinsung-1-Dong, Yangcheon Gu, Seoul, Korea.
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. §1734 solely to indicate this fact.
Address for reprint requests: C. D. Berdanier, Dept. of Foods and
Nutrition, Dawson Hall, Univ. of Georgia, Athens, GA 30602-3622.
Received 5 February 1999; accepted in final form 24 May
1999.
 |
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