From the Institute of Molecular Biology, University
of Oregon, Eugene, Oregon 97403, the
Unita' di Medicina
Molecolare, Ospedale Pediatrico "Bambino Gesu," Piazza S. Onofrio
4, 00165 Roma, Italy, and the ** Hospital for Sick Children, Toronto,
Ontario M5G 1X8, Canada
Received for publication, December 12, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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Cytochrome c oxidase (COX) deficiency
is the most common respiratory chain defect in childhood and is
clinically heterogeneous. We report a study of six patients with COX
deficiencies. Two of the patients had as yet undefined defects, three
patients had Surf-1 mutations, and one patient had a 15-base pair
deletion in the COX III subunit. We show that quantitative measurements of steady-state levels of subunits by monoclonal antibody reactivity, when used in combination with a discontinuous sucrose gradient methods,
provide an improved diagnosis of COX deficiencies by distinguishing
between kinetic, stability, and assembly defects. The two mutants of
undefined etiology had a full complement of subunits with one stable
and the other partially unstable to detergent solubilization. Both are
likely to carry mutations in nuclear-encoded subunits of the complex.
The three Surf-1 mutants and the COX III mutant each had reduced
steady-state levels of subunits but variable associations of the
residual subunits. This information, as well as aiding in diagnosis,
helps in understanding the genotype-phenotype relationships of COX
deficiencies and provides insight into the mechanism of assembly of the
enzyme complex.
Cytochrome c oxidase
(COX)1 is the terminal enzyme
complex of the respiratory chain. In eukaryotes, it is located in the
inner mitochondrial membrane where it catalyzes the transfer of
electrons from reduced cytochrome c to molecular oxygen (1).
This reaction is coupled with the translocation of protons across the
inner membrane, and the resulting electrochemical gradient is used to drive ATP synthesis and ion transport (2). Human COX is composed of 13 subunits, the three largest of which are encoded by mitochondrial DNA
(3). In addition to these 13 structural subunits, there are many
"assembly factors" required for the proper functioning of COX. In
yeast, over 30 different genetic complementation groups for COX
assembly have been reported (4, 5), and a number of genes involved in
the assembly of yeast COX have been identified (6-9). In humans, COX
assembly genes have been identified by several methods including
functional complementation of yeast mutants (10), homology searches of
the expressed sequence tags data base (11), and microcell-mediated
chromosome transfer (12, 13).
COX deficiency is the most commonly recognized respiratory chain defect
in childhood (14). The disease is clinically heterogeneous with
phenotypes including Leigh syndrome, hepatic failure, and myopathies
(12, 13, 15, 16). COX deficiency has been associated with mitochondrial
DNA mutations in COX I, II, and III (17-20), with large-scale
deletions of the mitochondrial genome (21) and with point mutations in
mitochondrial tRNA genes (22). There have also been reports of
autosomal inheritance of COX deficiency that have involved COX assembly
factors including Surf-1 (12, 13), Sco-1 (23), Sco-2 (24, 25), and
Cox10 (26). To date, there is no unequivocal evidence of a mutation in
the nuclear-encoded structural COX subunits causing human disease. One
patient with severe mitochondrial encephalomyopathy was reported to
have an altered Km for reduced cytochrome
c yet had no mutations in the mitochondrially encoded COX
subunits (27). The defect in this patient was presumed to have arisen
from a nuclear gene mutation, but no definitive data were provided.
With so many possible gene defects giving rise to COX deficiency, the
screening of patients is complicated, and a protocol that localizes the
possible mutations to groups of genes (e.g. mtDNA,
nuclear-encoded structural subunit genes, or genes for assembly
factors) would be useful. Recently the steady-state levels of
cytochrome c oxidase subunits in tissues from 17 patients
were examined by Western blotting using monoclonal antibodies made in
this laboratory (28). These patients showed a range of subunit profiles. Some had normal levels of all COX subunits and were classified as candidates for mutations in nuclear-encoded structural genes. Other patients, including one Surf-1 mutant, had reduced levels
of several of the subunits, which were classified as assembly mutants.
More recently, immunohistochemistry has been used to study a set of
COX-deficient patients, and similar conclusions were drawn (29). In
that study, patients showed either a reduction in all of the subunits,
none of the subunits, or just the mitochondrially encoded subunits.
Here, we have analyzed cytochrome c oxidase-deficient
patients by subunit composition as before (28), but we added an
analysis of patient mitochondria using sucrose gradient centrifugation. Our previous studies have established that this approach separates the
complexes of oxidative phosphorylation (OXPHOS) efficiently and,
in the case of complex I deficiencies, allows assembly of this complex
to be examined (30, 31). As we show, in combination, the data from the
two analyses improved the classification of cytochrome c
oxidase deficiencies and provided insight into both phenotype-genotype
relationships and assembly.
MRC5 Fibroblasts--
MRC5 fibroblasts were obtained from the
American Type Culture Collection (Manassas, VA). The population
doubling (PD) level of the cells was in the range of 35-45 at the time
of harvesting. All cells were grown as described before in high glucose
Dulbecco's modified Eagle's medium supplemented with 10% bovine calf
serum, 50 µg/ml uridine, 110 µg/ml pyruvate, and 10 mM
HEPES buffer to maximize growth rates (32). Rho0 MRC5
fibroblasts were derived by culturing MRC5 fibroblasts (PD = 28-30) continuously in permissive medium supplemented with 50 ng/ml
ethidium bromide for a further 16 population PDs.
Patient Cell Lines--
Primary fibroblast cultures of Patients
1, 2, 3, and 4 were obtained from needle skin biopsies performed during
the course of diagnostic evaluation at the Ospedale Pediatrico
"Bambino Gesu," Roma, Italy. The fibroblast culture of Patient 5 was obtained from the Hospital for Sick Children, Toronto, Ontario,
Canada. The 100% mutant cybrid cell line containing a 15-base pair
deletion of the COX III subunit (Patient 6) was kindly supplied by Dr. Michael King, Thomas Jefferson University, Philadelphia, PA. Specific details concerning this patient and the generation of the hybrid cell
line were described previously (19, 33)
Preparation of Mitochondria from Cell Lines--
Mitochondria
were prepared from ~5 × 107 cells by differential
centrifugation as described previously (31).
Activity Assays--
Cytochrome c oxidase activity
was measured basically as described previously (34). Briefly,
~80-100 µg of protein from digitonized fibroblasts or
homogenized muscle biopsy were added to a cuvette containing 1 ml final
volume of 10 mM potassium phosphate, pH 7.4, 1 mg/ml
cytochrome c reduced with sodium dithionite, and 0.025% of
lauryl maltoside. The oxidation of reduced cytochrome c at
550 nm was then followed spectrophotometrically (extinction coefficient
19.0 mM Immunoblot Analysis--
Proteins were separated on 10-22%
gradient SDS-polyacrylamide gels run according to Laemmli (35). Western
blotting was done as described previously (32), with the following
modifications. The proteins were transferred electrophoretically to an
0.45-mm polyvinylidine difluoride membrane in Towbin buffer. Reactive bands were detected using the ECL PlusTM detection reagent
(Amersham Pharmacia Biotech UK) and were imaged using the image
analyzer Storm 860 (Molecular Dynamics, Sunnyvale, CA). Fluorescence
was quantified using NIH Image and standardized to the loading control, porin.
The porin monoclonal antibody (Calbiochem, La Jolla, CA) was used at a
1:120,000 dilution in 5% milk/Ca2+- Mg2+-free
phosphate-buffered saline. The polyclonal Surf-1 antibody was a
kind gift from Dr. Eric Shoubridge, Montreal Neurological Hospital. All
other monoclonal antibodies used were prepared at the University of
Oregon and used in the following concentrations: complex I-39 kDa (2 µg/ml), complex II-30 kDa (5 µg/ml), complex II-70 kDa (0.01 µg/ml), complex III-core 2 (0.4 µg/ml), complex V- Sucrose Gradient Centrifugation--
Mitochondria (1 mg) were
resuspended at a protein concentration of 5 mg/ml in 1 mM
EDTA, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 1% lauryl maltoside, 100 mM Tris/HCl, pH 7.8. The mitochondria were incubated in this solution for
20 min on ice with stirring before insoluble membranes were pelleted by
centrifugation (10,000 × g, 10 min, 4 °C). The
supernatant (~200 µl) was layered on top of a discontinuous sucrose
gradient and centrifuged overnight at 4 °C (150,000 × g, 16.5 h, SW 50.1). The gradient was composed of 250 µl of 35% sucrose, 500 µl of 30% sucrose, 750 µl of 27.5%
sucrose, 1 ml of 25% sucrose, 1 ml of 20% sucrose, and 1 ml of 15%
sucrose. All sucrose solutions were prepared in 100 mM
Tris/HCl pH 7.8, 0.05% lauryl maltoside, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM
phenylmethylsulfonyl fluoride. The sucrose gradient was fractionated
from the bottom of the tube into 10 fractions, which were frozen at
Patient Characterization--
Cell lines from six patients
demonstrating different degrees of COX deficiency were studied.
The clinical presentations of these patients and specific
mutations that were identified are shown in Table
I. In muscle biopsy samples from Patients
1 and 2, the COX activity was drastically reduced by 77 and 76%,
respectively, compared with control values. On the other hand, the COX
activity in fibroblasts was much higher, with Patients 1 and 2 losing
only 30 and 20% of enzyme activity, respectively. It is not unusual for there to be tissue specificity of a defect when mtDNA mutations are
involved, because mtDNA replication and inheritance are stochastic in
somatic cells and can result in different mutational loads in different
tissues (36). However, muscle biopsies from Patients 1 and 2 were
screened for mutations in the mitochondrially encoded COX subunits I,
II, and III, and no mutations were found. The defects in these cell
lines are thus predicted to be nuclear-encoded. These two patients were
also screened for mutations in the COX assembly factors Surf-1, Sco-1,
and Sco-2, but again no mutations were found. The unusual tissue
specificity of these two mutants is discussed in more detail later.
In three of the patients (Patients 3, 4, and 5), we identified a SURF-1
mutation, resulting in undetectable levels of the Surf-1 protein (Fig.
1). Two of these are frameshift
mutations, and produce a premature translation termination. The third
(Patient 5) harbored a deletion between exons 1 and 2. Western blotting of Patient 5 using a different antibody from that used here has shown
the presence of some Surf-1 of altered
migration.2 Thus, the
mutation could result in an incorrectly spliced version of Surf-1.
Patient 4 presented with tubulopathy in addition to Leigh syndrome,
which is the most common clinical presentation associated with Surf-1
mutations. Patient 6 had a previously reported 15-base pair deletion in
COX III (19, 33).
Western Blot Analysis of Patient Cell Lines--
Western blot
analysis was performed on mitochondria isolated from all six patient
cell lines along with control MRC5 fibroblasts and MRC5 fibroblasts
that had been depleted of mitochondrial DNA (rho0).
Individual protein signals were normalized using the porin signal as a
control for equal loading. The signals of all proteins were set to
100% for the control MRC5 fibroblasts, and the levels of the
individual proteins in other cell lines are reported relative to this
value. The quantitative subunit profiles for each cell line
appear in Fig. 2. For Patients 1 and 2, all of the COX subunits probed were present at normal levels
(Fig. 2). Thus, the subunit profile alone does not provide diagnostic
information. The data for Patients 1 and 2 contrast with those of
Patients 3-6, each of which had a significant reduction in all of the
COX subunits probed. As shown in Fig. 2, the levels of subunits I, II,
IV, Va, and VIc were quantitatively similar in Patients 3 and 6, one a
Surf-1 mutant and the second a COX III mutant. The levels of subunits
in the other two Surf-1 mutants were different from Patient 3. Patient
5 had much more of subunits II and VIc, whereas Patient 4 showed
intermediate expression with more of subunits II and VIc than Patient 3 but less than Patient 5. Thus, steady-state levels alone do not
distinguish mtDNA from Surf-1 mutations.
Sucrose Gradient Analysis of Patient Cell Lines--
Mitochondria
from the six patient cell lines, control MRC5 fibroblasts, and
rho0 MRC5 fibroblasts were subjected to sucrose gradient
centrifugation. In the gradient, complexes are separated at a rate
depending largely on their size. The positions of complexes in the
gradient can then be determined by Western blotting of the fractions
with monoclonal antibodies directed against particular subunits. Ten
fractions were collected from each gradient, and Western blots were
performed on the fractions from each of the gradients using antibodies
to complex I-39 kDa subunit, complex V-
Fig. 4 shows the distribution of the COX subunits I, II, IV, and Va in
the gradient for each cell line. For the control MRC5 cells (Fig.
4A), all of these subunits peak in fractions 5 and 6, indicating that they are assembled in an intact complex.
Rho0 cells, on the other hand, show a much different
pattern (Fig. 4B). As subunits I and II are mitochondrially
encoded, they do not appear in the gradient. Subunits IV and Va are
present, and they both peak in fraction 8.
For Patient 1 with 30% reduction in COX activity in fibroblasts, not
only were all of the COX subunits expressed at near normal levels (Fig.
2), but the sucrose gradient shows a fully assembled complex (Fig.
4C). This patient may have a kinetic defect. Patient 2 had a
reduction of COX activity of 20%. In this case, there was evidence of
instability of the complex to detergent treatment, as a portion of
subunits IV and Va were found dissociated from the core complex (Fig.
4D).
Sucrose gradient evaluation of Patients 3, 4, and 5, each with
mutations in the COX assembly factor, Surf-1, confirmed that these are
all assembly mutants and showed that there are differences between them
in the amount and nature of the partially assembled complex (Fig. 4,
E-G). A fraction of subunits I, IV, Va, and all of the
residual subunit II present were found at the position of fully
assembled COX. Both Patients 3 and 4 retained significant COX activity
(see Table I), which is related to the amount of COX II present and
thereby the amount of assembled complexes. In both of these patients,
there were fractions containing subunits I + II + IV + Va, I + IV + Va,
and IV + Va. The third Surf-1 mutant (Patient 5) is different from the
other Surf-1 patients in regard to both steady-state levels of subunits
and in the assembly profile. This patient, with only 11% residual
activity, showed no fully assembled enzyme. However, there were
fractions containing I + II, I + Va, and IV + Va.
Patient 6, with a 15-base pair deletion in the COX III subunit, also
exhibited a severe assembly defect, with each of the four subunits
examined shifted in the gradient to lower molecular weight fractions
compared with controls (Fig. 4H). As in all the assembly
mutants, subcomplexes containing subunits I + II and IV + Va were detected.
COX deficiency presents with a plethora of phenotypes, which is
not surprising given the complexity of enzyme structure and the
multiple factors and many steps required for assembly of this, the
terminal oxidase of the respiratory chain (37). The challenge in
patient diagnosis is both to decide whether the condition is due to a
COX defect alone and to localize the possible mutations to a single or
limited number of genes. A functional COX requires three
mitochondrially encoded subunits, 10 nuclear-encoded subunits, some of
which are tissue specific, and an as yet unknown number of assembly
factors that includes Cox10, Sco-1, Sco-2, and Surf-1 (12, 13, 23-26).
Mutations of mtDNA include deletions that cause respiratory chain
deficiencies involving multiple respiratory chain complexes, including
COX and mutations in tRNA that cause MERRF (myoclonus epilepsy and
ragged red fibers) and MELAS (mitochondrial encephalomyopathy with
lactic acidosic and stroke-like episodes), which also have
defects in COX (36). Alternatively, there can be mutations in any of
the three mitochondrially encoded COX genes (for subunits I, II, and
III) as represented by Patient 6 in this study. An added complication
in understanding the genotype-phenotype relationships of mtDNA
mutations is the heteroplasmy, i.e. the presence of the
mutation in only a fraction of the thousands of copies of the
mitochondrial genome. The result is that cells contain a mixture of
normal and defective enzyme complexes giving a proportional activity
that can affect the functioning of different tissues differently,
i.e. the threshold effect (36).
Mutations of nuclear genes can occur in the structural subunits of the
COX complex (subunits IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, or
VIII), two of which (VIa and VIIa) have tissue-specific isoforms in
humans (34). If the incorporation of nuclear-encoded subunits into the
complex is an early event, defects due to these subunits could
affect the assembly, stability, and thus the steady-state levels of
enzyme or cause a change in the kinetics of enzyme turnover. Finally,
there can be mutations in any of a growing number of so called assembly
factors that are required to produce a functional COX. One of
these factors is Surf-1, represented in three patients in this study.
Thus, the routine sequencing of all the possible genes involved
will be laborious and even then not definitive until all the factors
required are identified.
In the meantime, analysis of the proteins has allowed some
differentiation of likely sites of mutation. The studies of von Kleist-Retzow et al. (28) and Rahman et al. (29)
show the variation in steady-state levels of COX subunits in COX
deficiencies, some of which are due to mtDNA defects, others undefined,
and one a Surf-1 mutant. This information provides some indication of
which defects allow enzyme assembly and which do not, based on the
ratios of subunits. However, the work was not done quantitatively, and
assembly was not examined directly. Another recent study has examined
several Surf-1 mutants by native blue gel electrophoresis, which, in principle, gives steady-state levels and assembly information (38). In this method, Tiranti and colleagues (38) were able to show
that, in the absence of Surf-1, there was some COX present in fully
assembled complexes along with partially assembled complexes. These
complexes contained subunit I, the only subunit examined in their study.
Here, we have conducted an analysis of subunit steady states based on
gel electrophoresis followed by Western blotting, but in a more
quantitative way than done previously. Samples from the same six
patients studied were then examined by sucrose gradient centrifugation,
which allows assembly of the enzyme complex to be evaluated. By adding
the sucrose gradient method, we are able to distinguish variants in
features of the COX complex not evident from steady-state levels of
subunits alone. A comparison of Patients 1 and 2 exemplifies
this point. Both patients have reduced cytochrome c
oxidase activity but normal levels of all the subunits. In Patient 1, the enzyme appears fully assembled, but in Patient 2, there is an
altered sedimentation in the gradient indicative of reduced stability
of the complex. The mtDNA of both patients was sequenced, and no
mutation was found. Therefore, both must have a mutation in a nuclear
gene. In the case of Patient 1, the most likely scenario is that there
is a mutation in one of the nuclear-encoded COX subunits that affects
enzyme turnover. One candidate is subunit Vb, mutations of which have
been found to alter enzyme kinetics in yeast (39). Other possibilities
are subunits IV and VIa, which regulate COX activity in response to ATP
binding (40, 41). A mutation in a nuclear-encoded COX subunit is also
the likely cause of the altered stability of the enzyme in Patient 2. The mutation could be in subunits IV or Va, which are released from the
complex by the detergent treatment and thus less tightly bound. Apart
from interacting with themselves, these two subunits interact mainly
with subunit I (3), so it is unlikely that mutation in other
nuclear-encoded subunits would have the observed effect. An interesting
feature of Patients 1 and 2 is that their levels of COX activity were
significantly lower in muscle biopsy (23 and 24% respectively)
than in fibroblasts in culture. This apparent tissue specificity may be
an added indicator of the locus of the mutation, perhaps pointing to
subunits VIa or VIIa, which have two isoforms in humans. Other
possibilities include mutations in as yet undefined assembly
factors/chaperones that have tissue specific isoforms. Additional
studies are ongoing with these two patients.
Four of the six patients examined here showed clearly altered assembly
of COX. This is already suggested for Patients 3, 4, and 6 by the
variable steady-state levels of each subunit. In the case of Patient 5, all subunits studied were present in equivalent amounts but at 50% of
wild type. The sucrose gradient analysis differentiates these assembly
mutants further and can resolve different mutations within the Surf-1
assembly factor by the patterns of subunit associations. In two of the
Surf-1 mutations there is an amount of fully assembled enzyme present,
which correlates with the amount of residual activity. All of the
subunit II is in this fraction. In addition, there are partial
assemblies of subunits. The third Surf-1 mutant, with higher levels of
subunits, is very different from the two described above. No
fully assembled enzyme is detected. Thus, either no COX was ever
properly assembled (although the cell lines have 11% residual COX
activity), and/or any complex that does assemble is much more labile
and disrupted by centrifugation in the detergent lauryl maltoside.
Patient 6, with a subunit III mutation, looks very much like Patients 3 and 4, who have defined Surf-1 mutations based on the steady-state levels of subunits, but is more like Patient 5 in having no fully assembled complex. As with Patient 5, the subunit profile on the sucrose gradient could result from failure of the enzyme to assemble, instability of the assembled enzyme, or both effects. However, it has
proved possible to remove subunit III from mammalian cytochrome c oxidase without disrupting the core of the enzyme complex
(subunits VIa, VIb, and VIIa are also lost) while retaining electron
transfer activity (42-44); this would argue that the mutation in COX
III affects assembly directly. It is likely then that COX III has some
chaperone-type role in assembly, in addition to any (as yet poorly
defined) functional role in the complex.
The sucrose gradient analyses of the six patients reveal at least two
partial assemblies of subunits. Subunits IV and Va remain associated in
Patients 2-6, in each case running together ahead of the position
expected of individual subunits. These two subunits are found in a
stable aggregate, even in enzyme missing the three mitochondrially
encoded subunits, i.e. in rho0 cells. It is
generally agreed that subunit I is inserted into the mitochondrial
membrane early in assembly with association of subunit II and binding
of hemes and copper atoms facilitated by the assembly factors
Surf-1, Sco-1, Sco-2, and Cox10 (23, 26, 38, 45, 46). Nuclear-encoded
subunits and COX III are thought to be added later in the assembly
process (45). It appears that COX IV and Va form a separate
subassembly, possibly with other subunits such as Vb or VIIb (not
studied here), and that the core part interacts in one step of assembly
with the preformed complex of nuclear-encoded subunits. In summary, we have extended the methodology currently available for studying COX
deficiencies by adding a sucrose gradient step to Western blotting
studies that define which subunits are present but not their assembly
state. The information provided by sucrose gradient centrifugation in
conjunction with monoclonal antibodies differentiates and
classifies COX mutants more finely than previously possible. Further
studies of COX deficiencies, as here, will aid in defining complementation groups of defects by protein rather than by DNA analysis. The data obtained as different mutants are examined should
also shed more light on the pathway of assembly of this complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
cm
1). Each reported value is the average of
2-5 independent measurements.
(4 µg/ml), COX II (2 µg/ml), COX Va (2 µg/ml), COX I (2 µg/ml), COX VIc (2 µg/ml), and COX IV (0.5 µg/ml).
80 °C. For the Western blotting, 20 µl of each fraction was
loaded per lane. For quantitation purposes, the levels of subunits
present in each fraction were expressed as a percentage of the darkest
intensity band for each antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Clinical, genetic, and biochemical characteristics of investigated
patients
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Fig. 1.
Western blot analysis of Surf-1 in the
COX-deficient patient cell lines in comparison to control MRC5
cells. The subunit of complex V (CV-alpha) was used
to control for equal loading in the lanes. The patient lines
were loaded in the order indicated, with C standing for the
control MRC5 fibroblasts. Surf-1 protein was undetectable in Patients
3, 4, and 6.
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Fig. 2.
Graphical representation of the levels of COX
subunits in the COX-deficient patient cell lines, rho0, and
control MRC 5 mitochondria. All subunits were set to 100% for the
control fibroblasts. All signals were normalized using porin as a
control for equal loading. Each bar is the average of 2-5
measurements.
, complex III-core 2, complex II-30 kDa subunit, and COX subunits I, II, IV, and Va. Fig.
3 shows a representative Western blot
from a sucrose gradient of mitochondria from Patient 4. For simplicity
of presentation, the data from each sucrose gradient are graphed by
setting the highest intensity signal for each antibody in each gradient
to 100% and expressing the levels of each subunit in the various
fractions as a percentage of this signal (Fig.
4). It is therefore important to note
that the absolute level of a particular subunit may be only a small
fraction of the level in control cells. However, the absolute
expression level of each subunit can be obtained by referring back to
Fig. 2. The distribution of the complex II-30 kDa subunit was monitored
in each experiment and always ran in fractions 6 and 7, confirming the
reproducibility of these gradient experiments (data not shown).
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Fig. 3.
A representative Western blot from a sucrose
gradient of mitochondria from Patient 4. Lanes 1-10
correspond to fractions 1-10 collected from the sucrose gradient. The
lane marked M contained the molecular weight
marker, and the lane marked C was the positive
control (MRC 5 mitochondria). The antibody mixture from top
to bottom consisted of COX I, complex II-30 kDa, COX II, COX
IV, and COX 5a.
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[in a new window]
Fig. 4.
Graphical representation of the
position of four COX subunits in sucrose gradients. Western blot
signals were quantitated, and for each antibody, the fraction with the
highest intensity signal was set to 100%. The relative amount of
signal found in the other fractions was then represented as a
percentage of this value. Shown are data from: A, control
MRC 5 fibroblasts; B, rho0 MRC 5 fibroblasts,;
C, Patient 1; D, Patient 2; E, Patient
3; F, Patient 4; G, Patient 5; and H,
Patient 6. COX I is represented in purple, COX II in
pink, Cox IV in yellow, and COX Va in
green.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL24526 (to R. A. C.) and by Ricerca Finalizzata, Italian Ministry of Health (30.3/RF 98.37).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.
§ These two authors contributed equally to the work reported here.
¶ Supported by National Institutes of Health Training Grant GM07759.
To whom correspondence should be addressed. Tel.: 541-346-5881;
Fax: 541-346-4854; E-mail: rcapaldi@oregon.uoregon.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M011162200
2 B. Robinson, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: COX, cytochrome c oxidase; PD, population doubling; mtDNA, mitochondrial DNA.
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REFERENCES |
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