From the Department of Biochemistry, School of Biology, Moscow State University, Moscow 119899, Russian Federation
Received for publication, October 23, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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The mammalian purified dispersed NADH-ubiquinone
oxidoreductase (Complex I) and the enzyme in inside-out
submitochondrial particles are known to be the slowly equilibrating
mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report
here the phenomenon of slow active/de-active transition in intact
mitochondria where the enzyme is located within its natural environment
being exposed to numerous mitochondrial matrix proteins. A simple
procedure for permeabilization of intact mitochondria by
channel-forming antibiotic alamethicin was worked out for the
"in situ" assay of Complex I activity.
Alamethicin-treated mitochondria catalyzed the rotenone-sensitive
NADH-quinone reductase reaction with exogenousely added NADH and
quinone-acceptor at the rates expected if the enzyme active sites would
be freely accessible for the substrates. The matrix proteins were
retained in alamethicin-treated mitochondria as judged by their high
rotenone-sensitive malate-cytochrome c reductase activity
in the presence of added NAD+. The sensitivity of Complex I
to N-ethylmaleimide and to the presence of Mg2+
was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of
alamethicin-treated mitochondria was sensitive to neither
N-ethylmaleimide nor Mg2+. After exposure to
elevated temperature (37 °C, the conditions known to induce
de-activation of Complex I) the enzyme activity became sensitive to the
sulfhydryl reagent and/or Mg2+. The sensitivity to both
inhibitors disappeared after brief exposure of the thermally
de-activated mitochondria with malate/glutamate, NAD+, and
cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in
intact mitochondria and discuss its possible physiological significance.
In mammalian mitochondria NADH-ubiquinone oxidoreductase (Complex
I, coupling Site 1, EC 1.6.99.3) functions as the main entry to the
respiratory chain. The enzyme has an extremely complex structure being
composed of more than 40 different subunits (1, 2). It contains
multiple distinct redox components (FMN, a number of iron-sulfur
clusters and tightly bound ubiquinones) operating in unknown sequence
of the reactions coupled with vectorial translocation of protons from
matrix to intermembraneous space. The functions of a vast majority of
the enzyme subunits are not known. Most of the recent studies on
Complex I and its simpler procaryotic counterparts (Type 1 NADH
dehydrogenases) have focused on their structure (1-4), iron-sulfur
clusters location (5, 6), possible mechanism of proton translocation
(7-9), and the comparative molecular biology of the enzyme
(10-12).
Very little is known about regulatory properties of Complex I. The
bovine heart enzyme shows very complex kinetic behavior when assayed in
either forward or reverse reactions. Following pioneering observations
of Estabrook and co-workers (13) it has been shown in this laboratory
that Complex I in inside-out submitochondrial particles
(SMP)1 or as purified
dispersed preparation always exists as functionally heterogeneous
mixture of two clearly distinct enzyme forms (states) (see Refs. 7 and
14 for reviews). One form (A) is fully capable of catalyzing the high
turnover rotenone-sensitive NADH-ubiquinone reductase reaction. The
other, which we operationally call as de-activated form (D) is unable
to transfer electrons to the quinone acceptor but is fully capable of
the reactions with artificial electron acceptors such as ferricyanide
or hexaammine ruthenium (III). In the absence of the substrates (NADH
and oxidized quinone) or in the presence of NADH and completely reduced
ubiquinone (no turnover is permitted) the spontaneous slowly
established equilibrium between A and D forms is greatly shifted to the
latter. The A to D transition has extremely high activation energy (270 kJ/mol) (15) and does not proceed at a significant rate at temperatures below 20 °C. The D to A transition needs slow activating turnover(s) in complete NADH-ubiquinone reductase reaction and it proceeds rather
rapidly at ambient temperature (t1/2 ~ 10 s
at 25 °C) (16). The rate of turnover-dependent D The significance of the slow A It can hardly be overemphasized that an unambiguous demonstration of
any regulatory property of Complex I in intact mitochondria is
extremely difficult because the inner mitochondrial membrane is not
permeable to NADH and respiration of mitochondria in the presence of
NAD+-linked substrates involves, besides Complex I itself,
obligatory operation of the dicarboxylate transport system, particular
dehydrogenase, and the downstream components of the respiratory chain.
It also worth noting that Complex I in intact mitochondria, at least
its matrix-protruding part, operates within rich protein environment which may or may not significantly affect the catalytic and/or regulatory properties of the enzyme.
The aim of the studies reported here was 2-fold. First, we searched for
a reliable experimental procedure for direct quantitative measurement
of the Complex I catalytic activity in sealed mitochondria. We have
used the channel-forming antibiotic alamethicin (20, 21) previously
employed for unmasking several ATP-dependent enzymatic
activities in sealed membraneous preparations and to reveal latent NADH
oxidase activity in intact mitochondria (22-24). The present article
shows that permeabilization of mitochondria by alamethicin provides a
valuable tool for measurement of the specific NADH oxidase and/or
NADH-quinone reductase activities in mitochondria. The second problem
we have addressed was to find out whether slow pseudo-reversible
Complex I A Rat Heart Mitochondria--
These were isolated from
trypsin-treated heart muscle (two hearts were handled for one
preparation) essentially as described by Jacobus and Saks (25). The
final precipitate of mitochondria was suspended in 0.3 M
sucrose, 10 mM Hepes, 0.2 mM EDTA (potassium salts, pH 7.4), and BSA (1 mg/ml) and stored in ice. The mitochondria oxidized malate/glutamate (5 mM each) in the reaction
mixture comprising 0.25 M sucrose, 10 mM
Tris/Cl Bovine Heart SMP--
SMP were prepared (15) and their
NADH oxidase was activated (26) as described. The uncoupled particles
(in the presence of gramicidin D, 0.2 µg/ml) catalyzed the
rotenone-sensitive (more than 99%) NADH oxidase reaction at the
average rate of 1 µmol/min/mg of protein at 22 °C, pH 8.0.
Complex I--
Complex I was purified according the standard
procedure (27). Its activity was determined at 38 °C in the reaction
mixture containing: 0.25 M sucrose, 50 mM
Tris/Cl (pH 8.0), 0.2 mM EDTA, BSA (1 mg/ml), 2.5 mM MgCl2, 5 mM NaN3,
100 µM NADH, and 100 µM ubiquinone-1
(Q1) after preincubation for 20 min with soybean phospholipids (2 mg/mg of Complex I).
Bovine Heart Mitochondrial Matrix Protein Fraction--
This was
prepared from the supernatant left after sonic treatment of bovine
heart mitochondria during SMP preparation. The supernatant (15 ml)
stored at The NADH Oxidase and NADH-quinone Reductase--
The activities
were assayed at 30 °C as a decrease of absorption at 340 nm with 200 µM NADH as the substrate (oxidase) or 200 µM NADH and 100 µM ubiquinone-1
(Q1) in the presence of 1.5 mM KCN (reductase).
The standard assay mixture contained: 0.25 M sucrose, 50 mM Tris/Cl The Malate-Cytochrome c Reductase--
This was assayed
following cytochrome c reduction at 550 nm in the presence
of 5 mM malate, 5 mM glutamate, 1.5 mM KCN, and 15 µM cytochrome c.
The hypotonic assay mixture contained: 10 mM
Tris/Cl The Malate Dehydrogenase Activity--
This activity was
determined as the rate of NADH oxidation in the reaction mixture
containing 20 mM potassium phosphate (pH 8.0), 0.2 mM EDTA, 5 µM rotenone, 150 µM
NADH, and 20 µM oxaloacetate. The mitochondrial
preparations (~20 mg of protein/ml) were solubilized at 0 °C by
Triton X-100 (1%, w/v, 20 min) and diluted 10 times in 0.25 M sucrose, 10 mM Hepes, 0.2 mM EDTA
(pH 7.4). Small samples (~2 µg of protein/ml) thus treated were
added to the reaction mixture and NADH oxidation was started by the
addition of oxaloacetate.
The Aspartate-2-oxoglutarate Transaminase Activity--
The
aspartate-2-oxoglutarate transaminase activity of the mitochondria
solubilized by Triton X-100 was determined as the rate of NADH
oxidation in the reaction mixture containing 20 mM
potassium phosphate (pH 8.0), 0.2 mM EDTA, 5 µM rotenone, 150 µM NADH, 0.1 mM 2-oxoglutarate, 0.1 mM aspartate and malate
dehydrogenase (1 unit/ml). About 50 µg of the mitochondrial protein
per ml was added to the assay mixture.
Permeabilized Mitochondria--
The following procedure based on
our experimental findings (see "Results") was employed to prepare
the mitochondrial preparation capable of the rotenone-sensitive
oxidation of externally added NADH. Intact mitochondria (10-20 mg/ml)
were diluted 20 times with the mixture comprising of 0.25 M
sucrose, 10 mM Hepes/KOH (pH 7.4), 0.2 mM EDTA,
BSA (1 mg/ml), 2.5 mM MgCl2, and alamethicin (40 µg/ml). The suspension was incubated at 20 °C for 5 min,
diluted 2.5 times with the same cold mixture containing no
MgCl2 and alamethicin, and centrifuged at 30,000 × g for 15 min. Precipitated mitochondria were suspended in
0.25 M sucrose, 50 mM Tris/Cl Protein Content--
The protein content was determined with
biuret reagent (28) using BSA as the standard.
NADH, NADPH, NAD+, EDTA, Tris, Hepes, BSA, malic acid,
glutamic acid, L-aspartate, 2-oxoglutarate, ADP,
Q1 (C-7956, Lot 117H32541), cytochrome c, and
NEM were from Sigma. Malate dehydrogenase was from "Reanal"
(Hungary). Alamethicin was a kind gift from Dr. S. Kotelevtzev
(Laboratory of Physico-chemical membranology, School of Biology, Moscow
State University).
Catalytic Activity of Complex I in Alamethicin-permeabilized
Mitochondria--
Intact rat heart mitochondria prepared by a mild
isolation procedure as compared with other reductase preparations were
used to study the effects of alamethicin on permeability of their inner membranes for the respiratory substrates. Table
I demonstrates that besides expected
uncoupling effect on
It was of interest to know whether the matrix proteins are retained in
alamethicin-treated mitochondria. This was verified by measuring
several enzymatic activities which requires the enzymes located in
matrix. Table II shows that
alamethicin-treated washed mitochondria lost their endogenous
NAD+ whereas the preparation significantly retained their
malate dehydrogenase and transaminase. The specific cytochrome
c reductase activity (0.08) in the presence of added
NAD+ was close to that found in the standard polarographic
experiments (0.130) at State 3 (see "Materials and Methods"). It
should be noted that the hypotonic reaction mixture was used for the
cytochrome c reductase activity assay to provide
accessibility of the inner membrane for added cytochrome c;
thus quantitative comparison of the NADH oxidase and cytochrome
c reductase activities is to be taken only as an
approximation. The specific activities of malate dehydrogenase and
transaminase were decreased in the alamethicin-treated preparations.
This was not unexpected, because alamethicin pore is not specific and
permeable for large cations and anions (29). Thus alamethicin induces
swelling of mitochondlia which can change the permeability of the inner
membrane and disruption of the outer membrane (30).
The results presented above showed that alamethicin-treated
mitochondria can be used for assay of the specific in situ
Complex I activity. It was of interest to compare the kinetic
properties of the mitochondrial enzyme which is exposed to a number of
matrix proteins with those previously reported for inside-out SMP (7). Fig. 2 shows the concentration dependence
of the initial reaction rates on ubiquinone homologue Q1
and NADH which were essentially the same as those for SMP. Pronounced
inhibition of the NADH-Q1 reductase activity at high
concentrations of Q1 (Fig. 2A) was variable and
dependent on the particular sample of the commercially obtained
quinone. This phenomenon presumably is due to some unidentified inhibitory contaminants and merits further investigation. It should be
noted, however, that with one particular sample of Q1 the
same kinetic behavior was always seen for inside-out SMP and
alamethicin-treated mitochondria. The standard kinetic parameters of
the rotenone-sensitive NADH-Q1 reductase activity for
alamethicin-treated mitochondria and those for inside-out SMP are
summarized in Table III. The catalytic turnover numbers for different preparations were calculated using the
values for the enzyme content determined as the minimal amount of
piericidin, the specific irreversible inhibitor of Complex I (31, 32),
needed to block the activities. The very close turnover numbers and
Km values thus obtained for mitochondria and SMP
preparations suggest that the enzyme in sealed mitochondria behaves as
its active sites would be freely accessible for the substrates and that
the matrix located proteins do not affect the catalytic activity of
Complex I. Slightly lower enzyme turnover number in rat heart
mitochondria may be due to species difference.
Complex I A The Effect of Matrix Proteins on Complex I A The activity of mitochondrial Complex I is of great importance for
cell physiology because the enzyme serves as the main collector of
reducing equivalents derived from Krebs cycle substrates and modulation
of the enzyme activity is expected to influence the energetic status of
any aerobic cell. Despite widespread interest in the functional state
of the enzyme at the cellular level the lack of simple and reliable
methods for the quantitative determination of its activity in intact
mitochondria greatly hampered the progress in several areas of
bioenergetics, especially in those of medical importance since a number
of diseases are believed to be associated with some defects in Complex
I (33, 34). It is a general practice in mitochondriology to correlate
respiratory activity in the presence of NAD-dependent
dehydrogenase-linked substrates with the catalytic activity of Complex
I. Depending on the particular tissue and/or on a number of factors
hard to control such as intactness of the mitochondrial membranes,
deficiency in nicotinamide nucleotides, specific activities of
dicarboxylate translocases and dehydrogenases, such correlation may or
may not be judicious. An obvious way to overcome an uncertainty in the
specific Complex I activity in mitochondria might be to use a detergent
to abolish the permeability barrier for NADH. However, the ubiquinone
reductase activity of Complex I has been shown to be extremely
sensitive to a number of lipophilic compounds including detergents
(35-37), as can be illustrated by strong inhibition of the enzyme by
Triton X-100 (36). The permeabilization of intact mitochondria by
alamethicin provides a simple procedure for reliable quantitative assay
of Complex I without any interference with the substrate translocases and dehydrogenases. Indeed, the kinetic parameters of fully active Complex I in sealed rat heart and bovine heart mitochondria as reported
here are very similar to those determined for inside-out SMP (Table
III). Another possible important application of alamethicin-induced permeabilization is its use for qualitative and quantitative
determination of a heterogenity of sealed membraneous preparations of
Complex I such as submitochondrial or sub-bacterial particles. No
stimulation of the NADH-Q1 reductase activity in SMP was
found (Table I), thus suggesting that no enzymatically active
right-side out particles are present in the preparations routinely used
in our laboratory (15). In contrast, considerable stimulation of the
uncoupled NADH oxidase reaction by alamethicin was found for the
preparations obtained from Paracoccus denitrificans
cells2 which is in accord
with the previously reported stimulation by bee venom of NADH oxidation
by P. denitrificans subbacterial particles (38). The
disadvantage of using alamethicin for the specific assay of Complex I
is obvious impossibility of measuring the enzyme activity in energized
mitochondria, thus possible
Two diagnostic tests: Mg2+ susceptibility at alkaline pH
and NEM sensitivity showed that mitochondrial proteins which are in direct contact with the matrix-exposed part of the enzyme do not protect Complex I against A
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
A transition is decreased at alkaline pH and/or in the presence of
bivalent cations (16). A-form is insensitive to the SH-reagents whereas
the D-form is specifically labeled and irreversibly inactivated by NEM
and other sulfhydryl group reagents (16, 17).
D transition for physiological
regulation of Complex I activity and consequently for general metabolism in mitochondria remained obscure as does existence of the
phenomenon itself in intact mitochondria. Some general speculative
proposal on the subject has been put forward (14, 18), although they
remain speculative because no evidence for the enzyme A
D
transition in situ were yet available. To our knowledge
there is only one report in the literature which suggests indirectly
that Ca2
sensitivity of the de-activated Complex I was
the reason for a decrease of respiration rate with
NAD+-linked substrates seen in intact liver mitochondria
after Ca2+ load (19).
D transition exist when the enzyme operates in the
natural matrix protein environment. Having succeeded in measurement of
Complex I activity in situ we were able to show that this
unique property is indeed an intrinsic feature of the enzyme in mitochondria.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 0.2 mM EDTA, and 6 mM
potassium phosphate (pH 8.0) at the average rate of 14 and 130 (respiratory control ratio of about 10) nanoatoms of oxygen per min per
mg of protein in the absence and presence of 200 µM ADP,
respectively, at 22 °C.
20 °C was thawed and diluted 2 times with cold water. 13 ml of 100 mM Tris/Cl
(pH 7.5) was added and
pH of the mixture was adjusted to 6.0 with acetic acid. The slightly
turbid mixture was centrifuged (30,000 × g, 30 min) to
remove residual membranes, pH of clear supernatant was adjusted to 8.0 with 1 N KOH and solid ammonium sulfate was added up to
70% saturation. The mixture was left on ice for 20 min, precipitated
protein was collected (30,000 × g, 30 min), suspended
in 2.5 ml of 10 mM Tris/Cl
(pH 8.0), and
dialyzed for 24 h against 1 liter of the same solution. The clear
soluble protein fraction thus obtained was stored in liquid nitrogen.
(pH 8.0), 0.2 mM
potassium EDTA, and the enzyme preparation (mitochondria or SMP (~10
µg of protein/ml).
(pH 8.0), 0.2 mM potassium EDTA and
mitochondria (~25 µg of protein/ml). All the activities throughout
the paper are expressed as micromoles of NADH oxidized per min per mg
of protein.
(pH
8.0), 0.2 mM EDTA, and BSA (10 mg/ml), and stored in ice during the experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Effect of alamethicin on several oxidoreductase activities catalysed by
the mitochondrial preparations different degree of resolution
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Fig. 1.
Effect of alamethicin on oxidation of NADH by
intact rat heart mitochondria. Mitochondria (30-40 µg of
protein/ml) were incubated for 1 min at 30 °C in the standard (3 ml)
spectrophotometric cuvette in the mixture containing: 0.25 M sucrose, 50 mM Tris/Cl (pH
8.0), 0.2 mM EDTA, 2.5 mM MgCl2,
and alamethicin. The NADH oxidase reaction (
) was started by the
addition of 200 µM NADH. The NADH-quinone reductase
reaction (
) was started by the addition of 200 µM NADH
and 100 µM Q1. 1.5 mM potassium
cyanide was present in the NADH-Q1 reductase assay mixture.
100% of the specific activities correspond to 1.0 and 0.45 µmol of
NADH oxidized per min/mg of protein for NADH oxidase and
NADH-Q1 reductase, respectively. NADH oxidase and
NADH-Q1 reductase were 99 and 93% sensitive to 2.5 µM rotenone, respectively.
Enzymatic activities of intact and alamethicine-treated rat heart
mitochondria
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Fig. 2.
Concentration dependence of
NADH-Q1 reductase reaction catalyzed by
alamethicin-permeabilized rat heart mitochondria. Mitochondria
were permeabilized and assayed as described under "Materials and
Methods." 200 µM NADH (A) and 50 µM Q1 (B) were present in the
standard assay mixture.
Kinetic parameters of NADH-Q1 reductase reaction catalyzed by
Complex I in sealed mitochondria and inside-out submitochondrial
particles
D Transition in Mitochondria--
The differences
in catalytic properties of A and D forms of Complex I summarized in the
Introduction provide at least two simple diagnostic criteria for their
relative content in any particular preparation: one is the sensitivity
of NADH-quinone reductase to the sulfhydryl group reagents; the other
is a lag-phase in the catalytic activity which is seen as inhibition of
the enzyme by divalent cations at alkaline pH. Both tests were employed
in further studies of alamethicin-treated mitochondria. Fig.
3 shows the effect of Mg2+ on
time course of NADH oxidation at different pH in thermally de-activated
mitochondria. At pH 7.0 about 40% inhibition of the reaction rate by
Mg2+ was observed. The inhibitory effect of
Mg2+ was significantly increased at pH 9.0. Note, that the
rates of NADH oxidation in the absence of Mg2+ were the
same at pH 7.0 and 9.0. These results are in accord with the previously
reported kinetics of NADH oxidation by the D-form of Complex I in SMP
(16). In the experiments depicted in Fig.
4 the sensitivity of NADH oxidation to
NEM was compared for thermally de-activated mitochondria (A) and the
same preparation prepulsed with NADH to reactivate the enzyme (B).
Thus, qualitatively, permeabilized mitochondria show both
characteristic features of the enzyme A
D transition. To get
further insight into quantitative characteristics of the enzyme
transition, we examined the time course of its irreversible inhibition
by NEM in mitochondria and SMP treated under various conditions (Fig.
5). At 20 °C activated SMP and intact
and permeabilized mitochondria as isolated were resistant to prolonged
(20-40 min) NEM treatment (closed circles and
squares, no detectable de-activation of the enzyme occurs at
this temperature). In contrast at 37 °C, the enzyme was rapidly (t1/2 ~ 2 min) inactivated by NEM in both
preparations (open circles and squares).
Remarkably, exactly the same rates of NEM-induced inactivation were
found for bovine heart SMP and rat heart mitochondria. It seemed
conceivable that the dramatic difference between the inactivation seen
at different temperatures might be due to the temperature dependence of
the sulfhydryl group(s) alkylation reaction. However, this was not the
case as evident from the experiments where the preparation was first
de-activated at 37 °C, and then treated with NEM at 20 °C: rapid
inhibition was observed (Fig. 5, closed triangles). Thus,
the time dependence of the inhibition by NEM at 37 °C was evidently
due to the time dependence of the enzyme de-activation. Moreover, when
mitochondria were partially de-activated by incubation at 37 °C for
limited periods of time as indicated on the abscissa in Fig.
5 and further treated with NEM at 20 °C for 2 min (the time needed
to inhibit completely de-activated enzyme at this temperature), the
points (open triangles) corresponding to the residual
activity thus revealed perfectly fit the curve characteristic for the
time-dependent inhibition by NEM at 37 °C.
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Fig. 3.
Effect of Mg2+ on Complex I
activity in permeabilized thermally de-activated rat heart
mitochondria. Alamethicin-permeabilized mitochondria (see
"Materials and Methods") were thermally de-activated (1.6 mg of
protein/ml, 15 min at 37 °C) and their NADH-Q1 reductase
activity was then assayed at pH 7.0 (A) and 9.0 (B). The reaction was started by the addition of 200 µM NADH (indicated by the arrows). 10 mM MgCl2 was present in the standard assay
mixture (see "Materials and Methods") where indicated.
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Fig. 4.
Inhibition of de-activated Complex I in
mitochondria by NEM. Mitochondria were de-activated as described
in the legend to Fig. 3, and preincubated in the standard
NADH-Q1 assay mixture containing 2 mM NEM
(where indicated) for 2 min (A) before the reaction was
initiated by the addition of 200 µM NADH (indicated by
the arrows). B, the same as A except
de-activated mitochondria were pulsed with 10 µM NADH (1 min) before preincubation in the assay mixture with NEM.
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Fig. 5.
Irreversible inhibition of Complex I in
mitochondria and submitochondrial particles by NEM as a function of
active/de-active enzyme transition. Permeabilized mitochondria
(1.6 mg/ml) were incubated at 37 °C ( ) or 20 °C (
) in the
presence of 2 mM NEM in the standard NADH-Q1
assay mixture (NADH and Q1 were omitted) for the time
indicated on the abscissa. Small aliquots were withdrawn
from the samples and their NADH-Q1 reductase activity was
determined in the presence of 200 µM NADH and 100 µM Q1. The same experiments were done at
37 °C (
) and 20 °C (
) for fully activated SMP.
,
mitochondria were first preincubated at 37 °C in the absence of NEM
for the times incubated on the abscissa, then the suspension
was cooled down to 20 °C, 2 mM NEM was added, incubation
was continued for 2 min, and the residual NADH-Q1 reductase
activity was assayed.
, SMP were first thermally de-activated
(37 °C, 15 min) and then treated with 2 mM NEM at
20 °C as described for
. 100% of the activities correspond to
0.45 and 0.7 µmol of NADH oxidized per min/mg of protein for
mitochondria and SMP, respectively.
D Transition in
SMP--
Another approach for modeling of in situ Complex I
A
D transition was to see the effect of crude matrix protein
fractions on the slow interconversions of the enzyme forms in
inside-out SMP. No effects of added matrix fraction (3.3 mg/ml) on the
thermally induced de-activation or on NADH-induced reactivation were
found. In contrast to NEM, oxidized glutathione (5 mM) had
no effect on the D-form of Complex I in SMP (nonenzymatic
thiol-disulfide exchange reaction) or in the presence of matrix
proteins (possible enzyme-catalyzed exchange reaction, the results are
not shown). When the steady-state oxidation of the Krebs cycle
substrates was reconstituted in the model system by the addition of
malate plus glutamate and NAD+, the "turnover
conditions" drastically prevented the enzyme de-activation even after
incubation of the samples at 37 °C as long as 20 min (Fig.
6).
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Fig. 6.
Protective effect of the enzyme turnover on
thermally induced de-activation of Complex I. Activated SMP (1 mg/ml) were incubated in 0.25 M sucrose, 50 mM
Tris/Cl (pH 8.0), 0.2 mM EDTA for the time
indicated at 0 °C (gray bars) or 37 °C
(black and open bars) and their NADH oxidase
activity was then assayed in the standard mixture at pH 9.0 in the
presence of 10 mM MgCl2 (to prevent rapid
activation of the enzyme during assay, see Fig. 3). Open
bars, matrix protein fraction (0.4 mg/ml), 2 mM
NAD+, 10 mM malate, and 10 mM
glutamate were added to the preincubation mixture.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
D transition and strengthen our hypothesis on the physiological relevance of this phenomenon. An
obvious question arises: what are the possible physiological conditions
which may lead to the de-activation of Complex I in vivo? It
seems unlikely that D-form is present in vivo when Complex I
catalyzes the steady-state NADH oxidation (Fig. 6). However, rapid
de-activation is expected under strong hypoxic or anoxic conditions
when the enzyme turnover is prohibited. An important point for the
discussion of such a scenario is that reactivation of the de-activated
enzyme is a very slow process in the presence of divalent cations. The
millimolar free Mg2+ (39) and variable high concentrations
of Ca2+ (19, 40) are present in the mitochondrial matrix.
Thus Complex I is expected to stay in D-form for a long time after
reoxygenation. In addition to the adverse effects that result from
de-energization of mitochondria during anoxia, further adverse effects
are anticipated following re-oxygenation because the D-form is unable
to transfer electrons to ubiquinone but can reduce oxygen directly
producing large amounts of superoxide. It is well established that
D-form by all the parameters studied so far (14) is equivalent to the rotenone-inhibited enzyme and rotenone was reported to activate superoxide generation by Complex I (41, 42). The experiments aimed to
prove or disprove our hypothesis on anoxia-induced de-activation of
Complex I in intact mitochondria are underway in our laboratory.
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ACKNOWLEDGEMENTS |
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We thank Irina Krysova and Alexandra Ushakova for valuable technical assistance.
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FOOTNOTES |
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* This work was supported in part by Russian Foundation for Fundamental Research Grant 99004-48082 (to A. D. V.), National Programme for Advanced Schools in Science Grant 00-15-97798, and Royal Swedish Academy of Science Collaborative Grant 12557 (to A. D. V.).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.
To whom correspondence should be addressed: Dept. of Biochemistry,
School of Biology, Moscow State University, 119899 Moscow, Russian
Federation. Tel.: 95-939-28-18; Fax: 95-939-39-55; E-mail: adv@biochem.bio.msu.su.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009661200
2 V. G. Grivennikova, N. V. Zakharova, and A. D. Vinogradov, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: SMP, submitochondrial particles; NEM, N-ethylmaleimide; BSA, bovine serum albumin; Q1, homologue of natural ubiquinone with 1 isoprenoid unit at position 5 of the 1,4-bensoquinoid ring.
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REFERENCES |
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