South Bend Center for Medical Education, Indiana University
School of Medicine, University of Notre Dame, Notre Dame, Indiana
46556
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INTRODUCTION |
THE PRESENCE OF GUANINE
NUCLEOTIDES in the matrix of mitochondria is uncontested,
although the mechanisms that account for their presence in the matrix
are not known. In previous work (8), we demonstrated that
radioactivity in the matrix pool of guanine nucleotides in heart
mitochondria arose exclusively from transport of radioactive GMP, GDP,
and/or GTP across the inner membrane, rather than arising via
conversion from inosine or adenine compounds, or via uptake and
synthesis from the salvage pathways using guanine or guanosine. The
addition of a single labeled form of GMP, GDP, or GTP to isolated heart
mitochondria resulted in the redistribution and equilibration of the
label among all three forms of the nucleotide. Thus the specific form
or forms of the nucleotide actually transported have not been determined.
To date, only two mechanisms for the transport of nucleotides across
the inner mitochondrial membrane have been characterized. Both are
involved in the transport of ATP and ADP (1, 2, 6). The
first is the ADP/ATP translocase, a major protein of the inner
membrane, which catalyzes the electrogenic, antiport exchange of ADP
and ATP across the membrane (for review, see Ref. 6). In
active mitochondria, the net activity of this carrier is to promote the
exchange of medium ADP for matrix ATP. This exchange is extraordinarily
rapid (ms) and has been well characterized. It is completely inhibited
from the medium side by atractyloside and from the matrix side by
bongkrekic acid (6). The second adenine nucleotide
carrier, at a much lower activity in the membrane than the first,
promotes the net uptake or loss of matrix ADP/ATP, typically in
exchange for inorganic phosphate (for review, see Refs. 1
and 2). This carrier, called the MgATP/phosphate exchange carrier, has
been characterized in liver and kidney mitochondria and is insensitive
to atractyloside and bongkrekic acid (1, 2). This latter
carrier is important in maintaining and regulating matrix adenine
pools, particularly during growth, and as such might be a prototype for
a carrier functioning in guanine nucleotide transport. However, neither
of the characterized adenine carriers is thought to have any role in
the transport of other nucleotides (1, 2, 6).
Prior research (8) demonstrated that the matrix pool of
guanine nucleotides arose from the uptake of one or more of the phosphorylated forms of guanine nucleotide. The first goal of this work
was to quantitate the distribution of label among the three
phosphorylated forms of guanine nucleotide under several conditions and
to correlate the label distribution to rates of label uptake. The
second goal of this work was to characterize the mechanisms of this
transport process. These goals were accomplished by using a vacuum
filtration method to measure the rate of uptake of labeled guanine
nucleotides into the matrix of isolated intact rat heart mitochondria
under a variety of conditions.
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EXPERIMENTAL PROCEDURES |
Isolation and incubation of heart mitochondria.
Tightly coupled mitochondria were isolated from hearts of control rats
(Harlan Sprague Dawley) exactly as described previously (9). Intactness was monitored with an oxygen electrode by
determining the respiratory control ratio using glutamate as the
substrate (9). Only mitochondria with respiratory control
values greater than six were used in the transport experiments.
Isolated heart mitochondria were incubated at 30°C in a medium
originally defined for mitochondrial translation experiments
(9) containing mitochondria at 4 mg protein/ml in (in mM)
25 MOPS buffer, pH 7.2, 90 KCl, 2 magnesium sulfate, 5 potassium
phosphate, 0.4 EGTA, 44 mannitol, 14 sucrose, BSA (1 mg/ml), 2-4
ADP or ATP, 20 glutamate, and 0.1 each of the 20 amino acids. Additions
and changes in this standard medium are outlined where appropriate.
HPLC analysis of purine nucleotides.
Purine nucleotides from isolated heart mitochondria were extracted,
identified, and quantitated from samples by reverse-phase HPLC over an
Alltech nucleotide/nucleoside column as described previously
(8). All guanine and adenine nucleotides are separated and
identified by this method (8).
Guanine nucleotide transport: vacuum filtration.
In earlier work, the method of medium and matrix separation via
centrifugation through cold silicone oil worked well to obtain sufficient samples for nucleotide analysis by HPLC (8).
However, this approach was not appropriate for rapid or small-volume
sampling when HPLC analysis was not necessary. To study transport
specifically, a vacuum filtration method was adopted as described by
Winkler et al. (13), which is similar to the technique
used by Aprille (1) to study atractyloside-insensitive
[14C]ATP transport in isolated liver mitochondria. In
this method, the labeled compound to be studied was added to the
incubation medium (described above but minus BSA) at 0°C. Isolated
heart mitochondria were then added and mixed rapidly, and a time
0 aliquot (0.1 ml) was removed. The sample was then incubated at
30°C, and aliquots were removed at other time points. Each aliquot
was filtered under vacuum through a 0.65-µm Millipore filter
prechilled by washing with 5 ml of 4°C saline immediately before
addition of the sample. The filter was then rapidly washed with 10 ml
of cold saline, removed, dissolved in Formula 963 or Aquassure
scintillation fluid (Packard), and counted in a liquid scintillation
counter. Uptake of radioactivity was plotted against time, and, where
necessary, the initial rate of uptake was calculated from the early
time points. The final equilibrated levels were determined for some experiments from later time points. The saline wash cleared the intermembrane space so that only matrix contents remained on the filter. In most experiments, [14C]sucrose was included in
the incubation to assess the washing procedure. The amount of sucrose
left on the filter was consistently quite low and indicated that >95%
of compounds in the intermembrane space was removed. This eliminated
the need to subtract the nucleotide contribution of the intermembrane space.
The concentration, form, and specific radioactivity of the nucleotides
studied in the vacuum filtration experiments were monitored by taking
aliquots of the sample directly to an equal volume of cold 10% TCA at
appropriate times. This sample was neutralized using AG 11 A8 resin
(Bio-Rad) and analyzed by HPLC as described previously
(8). Note that this sample gives the total (medium + matrix) concentration for the given nucleotide; however, the matrix
space represents only 0.4% of the total [1 µl matrix space/mg mitochondrial protein (8) and 4 mg mitochondrial
protein/ml], so these samples effectively show changes in the medium only.
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RESULTS |
Vacuum filtration.
The uptake of labeled guanine nucleotides into the matrix was
determined by separating the matrix contents from the medium using
rapid vacuum filtration. Two controls were used to verify that vacuum
filtration measured the uptake of soluble nucleotides into the matrix.
First, experiments were conducted with [3H]guanine
nucleotide phosphates in which the incubations were carried out
entirely on ice, blocking protein-mediated uptake but not simple
diffusion. No uptake was observed (data not shown). An additional
concern was that uptake might represent incorporation into
mitochondrial RNA. Because CTP and UTP were not present in the medium,
RNA synthesis did not seem likely but could occur using endogenous
levels of these nucleotides. To prove that the radioactivity taken into
the matrix was soluble, filter disks were washed with 5% TCA instead
of saline, breaking open the matrix and washing out the acid-soluble
components while precipitating the RNA onto the filters. Only
background radioactivity was observed on the disks in these
experiments, demonstrating that incorporation of guanine nucleotide
into RNA did not account for radioactivity on the disk.
Time course of equilibration, redistribution, and uptake of
[3H]GTP, [3H]GDP, and [3H]GMP
into isolated heart mitochondria.
Previous work (8) showed that the addition of guanine
nucleotide (labeled with 3H on the base) to isolated
mitochondria resulted in the distribution of that label among all three
phosphorylated forms of guanine nucleotide both within the
mitochondrial matrix and in the medium. Depending on the rate of
interconversion relative to the rate of transport, the uptake of label
may have been limited to a single form or may have been the sum of all
three forms of guanine nucleotide, each with its own kinetic constants.
Thus the first goal of this study was to try to distinguish the
individual role of each phosphorylated species of guanine nucleotide in
the transport process. This was accomplished by incubating isolated
heart mitochondria with [3H]GTP, [3H]GDP,
or [3H]GMP and determining the time course of label
interconversion to other forms (Fig. 1)
together with the time course of label uptake into the matrix (Fig.
2). These experiments were done in the
presence (Fig. 1, A-C, and Fig.
2A) and absence of 2 mM ATP (Fig. 1,
D-F, and Fig. 2B) to dramatically
alter the interconversion of label. When 0.1 mM [3H]GTP
was added to isolated heart mitochondria in the presence of ATP, the
label rapidly equilibrated such that the medium concentration of
[3H]GTP fell to 0.07 mM with the concomitant rise of
[3H]GDP to 0.03 mM. Only a trace amount of
[3H]GMP was formed. The uptake of matrix label under this
condition was linear at a rate of 1.9 pmol · mg
protein
1 · min
1 (Fig.
2A). If the label was added as 0.1 mM
[3H]GDP (Fig. 1B), ~5 min was required for
equilibration of the label, during which time the labeled GDP medium
level fell from 0.1 to 0.03 mM as it was converted to 0.07 mM
[3H]GTP. Again, only a trace amount of
[3H]GMP was formed. The matrix uptake of label after
addition of [3H]GDP was hyperbolic with an initial rate
of 12 pmol · mg
protein
1 · min
1 in the first 2 min,
decreasing after 5 min of incubation to 1.5 pmol · mg
protein
1 · min
1 (Fig.
2A). The initial rapid rate of uptake was 6 times higher than the rate observed during the first 2 min of incubation with [3H]GTP, indicating that GDP was a much better substrate
for transport than GTP. Furthermore, as the level of
[3H]GDP decreased and the level of [3H]GTP
increased, the rate of label uptake into the matrix decreased, again
suggesting that GDP was a much better substrate for transport than GTP.
In fact, using the value of 12 pmol · mg
protein
1 · min
1 as representing the
rate of transport from the 0.1 mM [3H]GDP
pool, we could predict the rate of transport from the
[3H]GDP pool when both [3H]GTP and
[3H]GDP pools were present. The predicted
rate of uptake from the 0.03 mM [3H]GDP pool that rapidly
formed after [3H]GTP addition (Fig. 1A) was
3.6 pmol · mg
protein
1 · min
1. Because the
observed rate of uptake after [3H]GTP addition was only
1.9 pmol · mg
protein
1 · min
1 (Fig.
2A), the data indicated that the 0.07 mM
[3H]GTP pool inhibited uptake from the smaller
[3H]GDP pool. One interpretation of these data was that
both [3H]GTP and [3H]GDP were
transported into the matrix by this process but that transport of GTP
was much slower. Thus, when GTP was present, some of the carriers were
occupied in a slower transport process.

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Fig. 1.
Interconversion of labeled guanine nucleotides in the
medium. Isolated heart mitochondria were incubated as described in
EXPERIMENTAL PROCEDURES with the indicated
labeled nucleotide (0.1 mM, 5 µCi/ml) as follows: A and
D: [3H]GTP in the presence (A) and
absence (D) of 2 mM ATP. B and E:
[3H]GDP in the presence (B) and absence
(E) of 2 mM ATP. C and F:
[3H]GMP in the presence (C) and absence
(F) of 2 mM ATP. At the indicated times, an aliquot of each
sample was removed and acidified with an equal volume of 10% TCA. Each
sample was centrifuged, and the supernatant was decanted and
neutralized as described previously (8). The labeled
compounds were separated by HPLC, identified by comparison to
standards, and quantitated by both ultraviolet absorption and detection
of radioactivity by an in-line scintillation counter (8).
The radioactivity in each peak was converted to a concentration by
dividing by the specific radioactivity of the added nucleotide. The sum
of the 3 forms at each time point in each panel totaled 0.1 mM as
expected. Results are from a representative experiment.
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Fig. 2.
Time course of uptake of [3H]GTP,
[3H]GDP, and [3H]GMP in the presence and
absence of ATP. Mitochondria were incubated as described in
EXPERIMENTAL PROCEDURES with the labeled
compound indicated (0.1 mM, 5 µCi/ml) in the presence (A)
and absence (B) of 2 mM ATP. Samples were removed at various
times from 0 to 30 min, and matrix uptake was quantitated by vacuum
filtration as described in EXPERIMENTAL
PROCEDURES. The radioactivity on the filter was corrected
for background and was converted to picomoles of nucleotide by dividing
by the specific radioactivity of the labeled nucleotide pool. Results
are presented as means and SE of nucleotide uptake per milligram of
mitochondrial protein from 5 experiments. In A, the data are
presented using two y-axis scales. The solid lines are
plotted against the scale on the left and allow differences
in uptake to be observed. The dashed lines represent the same data
plotted against the scale on the right, which is the same
scale as shown in B and allows the data in A and
B to be directly compared.
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The equilibrated levels of [3H]GTP and
[3H]GDP observed in Fig. 1, A and
B, reflected the energy charge of the isolated mitochondrial preparation. The medium ratio of GTP to GDP in Fig. 1, A and
B, was 2.3, and the guanine energy charge was 0.85. These
data agreed exactly with the ATP-to-ADP ratio and adenine energy charge
determined for these same samples (data not shown). In other studies
from our laboratory in which the medium ATP-to-ADP ratio varied over a
wide range, the medium GTP-to-GDP ratio was always nearly identical. These data demonstrated that the adenine and guanine pools in the
medium were in rapid equilibrium, presumably mediated by an active
nucleoside-diphosphokinase known to be present in the intermembrane space (14).
In normal respiring mitochondria, the energy charge of the system is
maintained by ATP, which is in turn maintained by oxidative phosphorylation. Thus omission of ATP from the incubation medium should
drastically lower the predicted energy charge. When
[3H]GTP was added in the absence of ATP (Fig.
1D), it was steadily converted to [3H]GDP,
yielding final levels of 0.01 mM [3H]GTP, 0.080 mM
[3H]GDP, and 0.010 mM [3H]GMP, for a
[3H]GTP-to-[3H]GDP ratio of 0.14 and an
energy charge of 0.5, dramatically lowered from Fig. 1, A
and B. The matrix uptake of label under this condition was
slightly hyperbolic, rising to 14 pmol · mg protein
1 · min
1 at 5 min after
addition and falling to a fairly linear rate of 9.7 pmol · mg
protein
1 · min
1 from 10-30
min. This rate was over five times faster than that observed in the
presence of ATP (Fig. 2, compare A, dashed line, with
B). When label was added as
[3H]GDP in the absence of ATP (Fig.
1E), it was only slowly phosphorylated to
[3H]GTP, presumably by substrate level phosphorylation
via the succinate thiokinase step of the Krebs cycle. After 30 min of
incubation, the level of [3H]GDP fell gradually from 0.1 to 0.08 mM with concomitant increases in [3H]GTP and
[3H]GMP of 0.01 mM levels each, the same values observed
for [3H]GTP addition in Fig. 1D. As above, in
the presence of ATP, the guanine nucleotide levels distributed to
reflect the energy charge of the isolated mitochondrial system. The
rate of matrix uptake of label after addition of [3H]GDP
in the absence of ATP was hyperbolic with an initial 2-min rate
of 78 pmol · mg
protein
1 · min
1. This rate was 6.5 times faster than the rate of label uptake observed when
[3H]GDP was added in the presence of 2 mM ATP (Fig. 2,
compare A, dashed line, with B). Because
[3H]GDP was essentially the only labeled form present
during the rapid uptake phase (0-5 min), the actual species of
nucleotide transported across the inner membrane must have been
[3H]GDP and the uptake rate represented uptake solely
from the [3H]GDP pool. As noted earlier, this value could
be used to calculate uptake rates from the [3H]GDP pool
when both labeled GDP and GTP pools were present, allowing an
estimation of the potential role of the [3H]GTP pool. The
data in Fig. 1D showed that, after addition of 0.1 mM
[3H]GTP, the [3H]GDP concentration was
already at 0.012 mM immediately after addition (0 time point), rising
to 0.054 mM by 5 min. At the initial concentration of
[3H]GDP, the predicted rate of label uptake from the
[3H]GDP pool alone would be 9.4 rising to as much as 38 pmol · mg protein
1 · min
1
at 5 min. The observed rate of uptake after [3H]GTP
addition noted above (Fig. 2B) was 7.2, rising to 14 pmol · mg protein
1 · min
1
at 5 min. Because the observed rate of uptake potentially from both the
[3H]GTP and [3H]GDP pools was less than
that expected from the [3H]GDP pool alone, these data
suggested that [3H]GTP inhibited the transport of
[3H]GDP. Thus the data obtained here in the absence of
ATP, as well as the data obtained above in the presence of ATP,
suggested that GDP was markedly preferred by this transport system,
whereas the presence of GTP inhibited uptake of GDP. As noted above,
this inhibition was probably caused by the much slower transport of GTP
occupying the carrier.
The hyperbolic shape of the label uptake curve after addition of
[3H]GDP was attributed to approaching equilibration of
[3H]GDP transport. The 0.1 mM medium level of
[3H]GDP was equivalent to a matrix level of 100 pmol
[3H]GDP/mg mitochondrial protein [assuming a 1 µl/mg
protein space (8)]. Thus the 400 pmol/mg protein value
reached after 30 min of transport represented a fourfold concentration
of label in the matrix. This fourfold increase in concentration
determined by label was also confirmed by HPLC chemical measurement of
GDP in the medium and matrix (data not shown). These data were very similar to the active compartmentalization of adenine nucleotides observed in isolated heart mitochondria (McKee and Bentley, unpublished observations) and suggested that, in the absence of ATP, heart mitochondria concentrate guanine nucleotides in the matrix in much the
same way as they do adenine nucleotides. However, in the presence of
ATP (Fig. 2A), the matrix uptake of guanine nucleotides (0.1 mM) after 30 min of incubation was still significantly below the medium concentration.
When label was added as [3H]GMP (Fig. 1, C and
F), the label did not readily equilibrate with the other
phosphorylated forms and showed only a slow conversion to GDP and GTP.
Although slow, this conversion nevertheless occurred at a constant rate
of 13% in 30 min (0.11 nmol · min
1 · mg
1
mitochondrial protein) in the presence of ATP (C) and fell
to just 1.4% in 30 min in the absence of ATP (F). This
steady conversion suggested the presence of a low-activity nucleoside
monophosphate kinase capable of using GMP as a substrate. To our
knowledge, this activity has not been previously shown in mitochondria.
The subcellular localization of the three known members of the
mammalian guanylate kinase family has not been completely examined, but there is no evidence that any of them reside in the mitochondria (3, 13). Alternatively, GMP may be a weak substrate for
the mitochondrial matrix form of adenylate kinase, which is a GTP:AMP phosphotransferase (4). The 10-fold decrease in conversion observed on omitting ATP (compare C and F, Fig.
1) points out the expected role of ATP or GTP as the donor phosphate
for this reaction.
The rate of uptake of [3H]GMP in the presence or absence
of ATP was very similar (Fig. 2, compare A, dashed line, and
B) and analogous to the rate observed when
[3H]GTP was added in the presence of ATP (Fig.
2A, circles vs. squares). Because label was present almost
solely as [3H]GMP (Fig. 1, C and
F), particularly during the first 10 min of incubation,
these data clearly demonstrated that [3H]GMP was
transported across the inner membrane. Furthermore, HPLC analysis of
the matrix contents after a 60-min incubation with 0.1 mM
[3H]GMP indicated that [3H]GMP was also the
predominant form of guanine nucleotide in the matrix (>90%, data not shown).
Effect of atractyloside on guanine nucleotide uptake.
The data above described a transport process that preferred GDP, was
stimulated by the omission of ATP, and was capable of concentrating
guanine nucleotide in the matrix in the absence of ATP. A good
candidate for such transport was the ADP/ATP translocase, an exchange
carrier that typically transports ADP into the matrix in exchange for
ATP. GDP, and perhaps GTP, might serve as secondary substrates for this
carrier in the heart, in which case their transport would be
competitively inhibited by the presence of ATP. Because this carrier is
specifically inhibited by atractyloside (6), we determined
if guanine nucleotide served as a substrate for this translocase by
testing the effects of atractyloside on guanine nucleotide uptake. This
was done under two conditions. The first used a very low exogenous
concentration of [3H]GTP in which 5 µCi/ml label was
added directly from the commercially prepared isotope (10 Ci/mmol = 0.0005 mM GTP) (Fig. 3A).
The second condition (Fig. 3B) used a more physiological
concentration of [3H]GTP (0.5 mM, 0.02 mCi/ml). These two
conditions were chosen because preliminary evidence suggested that the
characteristics of transport at these two widely different
concentrations were significantly different, a fact confirmed by the
data in Fig. 3. At low exogenous concentrations of
[3H]GTP (Fig. 3A), the label equilibrated
between the medium and the matrix within 30 min to a level that was
concentrated two- to threefold in the matrix [assumes 1 µl/mg volume
in the matrix (8)]. Because this did not occur at 4°C,
carrier-mediated uptake of the label was clearly demonstrated.
Furthermore, the presence or absence of ATP or of atractyloside had
only a minimal effect on uptake rates and equilibration levels,
although equilibrium levels of guanine nucleotides in the absence of
ATP and atractyloside did show a modest statistical difference from
levels in the presence of both (P < 0.001). Note that
at this low added level of guanine nucleotide, the endogenous amount of
guanine nucleotide [~4 nmol guanine nucleotide/ml (8)]
was eightfold higher than the amount of GTP added. Because the extent
to which this initial matrix pool enters and dilutes the medium pool of
guanine nucleotides was not known, the concentration and specific
radioactivity of GTP in the medium were not known. As a result, the
data in Fig. 3A are expressed simply in units of
radioactivity taken up into the matrix. In contrast, at the more
physiological concentration of [3H]GTP, the endogenous
GTP was negligible and the data are expressed as nanomoles of GTP
uptake per milligram of mitochondrial protein (Fig. 3B). At
a physiological level of GTP, the effect of ATP and atractyloside on
the uptake of label was dramatic (Fig. 3B). In the absence
of ATP and atractyloside, uptake of label added as
[3H]GTP was rapid and hyperbolic. The rate at the first
time point (15 min) was 33 ± 4.2 pmol · mg
protein
1 · min
1, and the level as
equilibration approached was 1,500 pmol/mg protein, a level threefold
above the medium level (Fig. 3B, inverted triangles). The
addition of 2 mM ATP to the incubation inhibited uptake by 90% (Fig.
3B, triangles, P < 0.001), whereas the
addition of atractyloside inhibited uptake by 98% (Fig. 3B,
squares, P < 0.001). Dose-response experiments with
atractyloside have shown that the concentration of atractyloside used
in these studies (1 µM) provided maximal inhibition of uptake of both
labeled guanine and adenine nucleotides (data not shown). Increasing
the concentration of atractyloside had no further affect on uptake.
These data clearly implicated the ADP/ATP translocase in the transport
of guanine nucleotide but raised the question as to the physiological
significance of this transport, given the competitive inhibition of
guanine nucleotide transport by ATP. The data in Fig. 3B
suggested that 2 mM ATP was not quite sufficient to completely inhibit
guanine nucleotide uptake over this carrier because addition of
atractyloside to the 2 mM ATP resulted in a further modest inhibition
(3.3 ± 0.2 to 2.4 ± 0.2 pmol uptake · mg
protein
1 · min
1 for results with
ATP and without atractyloside vs. with both ATP and atractyloside,
P < 0.001, Fig. 3B).

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Fig. 3.
Effect of atractyloside (Atr) and ATP on uptake of
[3H]GTP. Mitochondria were incubated as described in
EXPERIMENTAL PROCEDURES with 2 different
concentrations of exogenous [3H]GTP: 5 µCi/ml at 10 Ci/mmol (0.0005 mM) in A and 0.5 mM, 5 µCi/ml in
B. A: uptake of [3H]GTP was
measured as described in the legend of Fig. 2 except that the uptake of
label was not divided by the specific radioactivity (see text) and is
expressed as dpm taken up by the matrix per milligram of mitochondrial
protein. The level of radioactive GTP in the matrix that would match
the medium radioactive concentration is indicated (11,000 dpm/µl,
dashed line), which assumes a matrix space of 1 µl matrix water/mg
protein (8). B: uptake of GTP is expressed as
described in EXPERIMENTAL PROCEDURES as nmol of
GTP taken up per milligram of mitochondrial protein. The matrix
concentration of GTP equivalent to the 0.5 mM medium concentration is
0.5 nmol GTP/mg and is shown as a dashed line. Uptake of GTP in both
panels was measured under 4 conditions: , +2 mM ATP and
+1 µM atractylate; , +2 mM ATP and no atractylate;
, no ATP and +1 µM atractylate; , no
ATP and no atractylate. Results represent means and SE of 3-6
independent determinations.
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To better understand the competitive nature of ATP in the uptake of
guanine nucleotides, rates of label uptake at a fixed addition of 0.5 mM [3H]GTP were measured as a function of increasing
medium ATP concentrations (Fig. 4).
Half-maximal inhibition of label uptake was observed at 0.15 ± 0.04 mM ATP, and maximum inhibition was observed at ATP levels
4 mM.
As expected, at maximal inhibition, the rate of uptake became the same
as the rate observed in the presence of atractyloside. The level of
maximum inhibition of [3H]GTP uptake was reached at an
8-to-1 ratio of adenine nucleotide to guanine nucleotide. These data
explained the lack of affect of atractyloside on [3H]GTP
uptake at very low exogenous [3H]GTP levels (Fig.
3A) because, in the absence of addition of exogenous
nucleotides, the adenine nucleotide level was about eight times greater
than the guanine nucleotide level (8). Thus uptake of
label when [3H]GTP was added at the low concentration
(Fig. 3A) was already restricted to the
atractyloside-insensitive carrier. Because the ratio of adenine
nucleotide to guanine nucleotide under typical conditions in the cell
is ~14:1 (5, 10), it seems unlikely that guanine
nucleotides are carried on the ATP/ADP translocase under physiological
conditions, suggesting a role for the slow atractyloside-insensitive
transport process. Interestingly, at physiological levels of GTP, the
addition of 2 mM ATP to atractyloside stimulated the uptake of label
added as [3H]GTP over twofold (1.0 ± 0.2 to
2.4 ± 0.2 pmol uptake · mg
protein
1 · min
1 for experiments
without ATP and with atractyloside vs. those with both ATP and
atractyloside, respectively, P < 0.001, Fig. 3B). This suggested that ATP did not compete for transport
on the slower atractyloside-insensitive carrier.

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Fig. 4.
Dose-response curve of ATP concentration ([ATP]) on
[3H]GTP uptake in the presence and absence of
atractyloside. Mitochondria were incubated with [3H]GTP
(0.5 mM, 10 µCi/ml) as described in the legend to Fig. 2, in the
presence and absence of 1 µM atractylate at various concentrations of
ATP as indicated. The initial rate of GTP uptake per minute per
milligram of mitochondrial protein (velocity) was calculated for each
concentration of ATP by obtaining the best-fit slope to the linear
portion of the uptake time course by linear regression (SigmaPlot 5.0, SPSS). The velocity obtained was plotted against the ATP concentration.
Results represent means and SE of 3 independent determinations.
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Specificity of transport on the atractyloside-insensitive carrier.
The slow atractyloside-insensitive pathway of uptake was detected in
Figs. 3 and 4 using [3H]GTP as the added label. However,
as noted in Fig. 1, A and D, label added as
[3H]GTP was converted partially to [3H]GDP,
making it difficult to know which species (or both) was actually
transported across the inner membrane on this slower carrier.
Furthermore, because the uptake of [3H]GMP was not
reduced by the addition of ATP (Fig. 2), it seemed likely that uptake
of [3H]GMP occurred via the atractyloside-insensitive
pathway. To obtain a better understanding of the specificity of guanine
nucleotide uptake via the atractyloside-insensitive pathway, the uptake
of [3H]GMP, [3H]GDP, [3H]GTP,
and [3H]IMP were determined in the presence of 1 µM
atractyloside and 2 mM ATP and compared (Fig.
5). The distribution of label observed for each addition of guanine nucleotide was essentially the same as
shown in Fig. 1, A-C, except that
atractyloside somewhat slowed the interconversion process (data not
shown). Label added as [3H]IMP remained solely in IMP.
Because the rate of label redistribution was slower in the presence of
atractyloside, a preference for a specific species of guanine
nucleotide for this transport process should be readily apparent at the
early time points. However, the uptake of label when added as
[3H]GMP, [3H]GDP, or
[3H]GTP was about the same regardless of the form of
guanine nucleotide added (Fig. 5, solid lines). Thus the
atractyloside-insensitive transport pathway appeared to move all three
nucleotides more or less equally. This differed from the uptake results
observed in the absence of atractyloside (Fig. 5, dashed lines), which demonstrated that there was a clear preference for uptake of
[3H]GDP, a preference now ascribed to the ADP/ATP
translocase. The similarity of uptake rates of [3H]GMP in
the presence and absence of atractyloside (Fig. 5, dashed vs. solid
line) demonstrated that GMP was not a substrate for the ADT/ATP
translocase and was transported solely via the insensitive pathway. As
an additional test of specificity, the transport of a closely related
nucleotide, [3H]IMP, was measured (Fig. 5), and no uptake
of label into the matrix was observed, demonstrating that this pathway
has specificity for guanine nucleotide. In earlier work, we
demonstrated that guanine and guanosine were also not transported into
the matrix (8). The failure of transport of IMP, guanine, and guanosine under these conditions provided strong evidence that the slow uptake of
guanine nucleotide by this mechanism was not an artifact caused by a
small number of damaged mitochondria in the preparation.

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Fig. 5.
Time course of uptake of [3H]GTP,
[3H]GDP, [3H]GMP, and [3H]IMP
on the atractyloside-insensitive carrier. Mitochondria were incubated
as described in the legend of Fig. 2 except that 2 mM ATP was present
in all samples, and uptake was measured in the presence of 1 µM
atractylate (solid lines). Samples were removed at various times from 0 to 60 min, and matrix uptake was quantitated by vacuum filtration and
expressed as described in the legend of Fig. 2. Guanine nucleotide
results are presented as means and SE of nucleotide uptake per
milligram of mitochondrial protein from at least 4 experiments; IMP
results are the means of 3 experiments. The uptake curves of
[3H]GMP, [3H]GDP, and [3H]GTP
from Fig. 2 (no atractylate) are shown as dashed lines for
comparison.
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Effect of nucleoside/nucleotide transport inhibitors on
[3H]GTP uptake.
The thiol active reagents mersalyl and N-ethylmaleimide have
been shown to inhibit the MgATP/phosphate exchange carrier
(2), whereas p-hydroxymercuribenzoic acid has
been shown to inhibit uptake of deoxyguanosine, all in liver
mitochondria (12). The effect of these three reagents on
the atractyloside-insensitive transport of GTP was examined. Mersalyl
(0.01-0.2 mM), N-ethylmaleimide (0.2-1 mM), and
p-hydroxymercuribenzoic acid (0.1 mM) were added to isolated
heart mitochondria in the presence of 2 mM ATP, atractyloside, and 0.1 mM [3H]GTP, and uptake of label was determined. These
reagents, at concentrations that have been reported to maximally
inhibit the carriers above (2, 12), had no effect on the
uptake of labeled GTP (data not shown). In the presence of a range of
mersalyl concentrations, GTP uptake was recorded only during the first
5 min of incubation, as this reagent appeared to damage the
mitochondria, as evidenced by their aggregation. These data suggested
that an active sulfhydryl group was not involved with the
atractyloside-insensitive transport of GTP. Nitrobenzylmercaptopurine
ribonucleoside (NBMPR) has been shown to inhibit nucleoside/nucleotide
transport in nuclei and across plasma membranes (7).
NBMPR, at a concentration reported to maximally inhibit this process
(0.01 mM), was tested in the same manner as the reagents above and was
also without effect on GTP uptake.
Effect of incubation temperature on [3H]GTP uptake.
As shown in Fig. 6, the rate of uptake of
[3H]GTP in the presence and absence of atractyloside was
dependent on the temperature of incubation as expected for a
protein-mediated process. These data provide evidence that the slow
uptake of guanine nucleotide is not mediated by simple diffusion or
diffusion through some type of channel.

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Fig. 6.
Velocity of GTP uptake vs. temperature. Mitochondria were
incubated at 4 different temperatures, using [3H]GTP at
0.01 mCi/ml, 0.1 mM in the presence of 2.0 mM ATP, and in the presence
or absence of 1 µM atractylate. Aliquots were removed at various
times from 0 to 60 min, and matrix uptake was quantitated by vacuum
filtration as described in the legend of Fig. 2. The rate of uptake
(velocity) was calculated for each concentration of GTP as described in
the legend of Fig. 4 and plotted as a function of temperature. The
symbols represent means and SE of the velocity of uptake of GTP of 4 experiments.
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Kinetic constants of the atractyloside-sensitive and -insensitive
guanine nucleotide carriers.
The data in Figs. 1 and 2 indicate that the atractyloside-sensitive
carrier displayed a marked preference for GDP. The kinetic constants of
this uptake process were determined by measuring the velocity of label
uptake measured at a range of GDP concentrations in the absence of ATP
(Fig. 7,
top). As shown in Fig. 2B, the concentration of
labeled GDP remained relatively constant over the time course
(0-15 min) used to measure initial velocity. Kinetic constants of
[3H]GDP uptake were determined by computing the best fit
of these data (SigmaPlot 5.0, SPSS) to the transport equation for
carrier-mediated uptake: V = Vmax[GDP]/(Km + [GDP]), where Km is the Michaelis-Menten constant and V is velocity. As shown in Fig. 7,
top, an excellent fit of the data was obtained,
demonstrating the presence of a saturable carrier protein with a
Vmax of uptake of 946 ± 53 pmol · mg protein
1 · min
1
and a Km of 2.9 ± 0.3 mM for GDP. At
equilibrium, this carrier was capable of concentrating label in the
matrix about fourfold over the medium for GDP additions below 0.25 mM,
a fact confirmed by HPLC analysis. However, as GDP levels increased,
the ratio of matrix to medium GDP concentrations decreased to 2 at 1 mM GDP and to 1 at 5 mM GDP.

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Fig. 7.
Kinetic values of guanine nucleotide uptake on
the atractyloside-sensitive and -insensitive carriers. Top:
[3H]GDP uptake on the atractyloside-sensitive carrier.
Mitochondria were incubated as described in the legend of Fig. 2 in the
absence of ATP and with varying concentrations of
[3H]GDP. Aliquots were removed at various times from 0 to
30 min, and matrix uptake was quantitated by vacuum filtration as
described in EXPERIMENTAL PROCEDURES. The
initial rate of uptake (velocity) was calculated over the first 15 min
for each concentration of GDP by obtaining the best-fit slope to the
linear portion of the uptake time course by linear regression
(SigmaPlot 5.0) Velocities were plotted as a function of concentration.
Points with error bars represent the mean and SE of 5 independent
experiments; other points represent the average of 2 independent
experiments. The best-fit line to the data was calculated by computer
using the equation V = Vmax[GDP]/(Km + [GDP]) (SigmaPlot 5.0). The best fit was obtained with
Vmax = 946 ± 53 pmol · mg 1 · min 1,
Km = 2.9 ± 0.3 mM, and
r2 = 0.998. Middle:
[3H]GTP/GDP uptake on the atractyloside-insensitive
carrier. Mitochondria were incubated as described in the legend of Fig.
2 with 2 mM ATP and 1 µM atractylate present in all samples with
varying concentrations of [3H]GTP. The data were obtained
and presented as described for top. The symbols represent
means and SE of the velocity of uptake of GTP for 5 experiments. The
best-fit line to the data points for the carrier-mediated uptake curve
was calculated by computer using the equation V = Vmax[GTP]/(Km + [GTP]) (SigmaPlot 5.0). A good fit was obtained between 0.025 to 25 mM GTP with Vmax = 48.7 ± 1.4 pmol · mg 1 · min 1,
Km = 4.4 ± 0.4 mM, and
r2 = 0.996. The predicted line
underestimates the rates below 0.01 mM. Bottom:
[3H]GMP uptake on the atractyloside-insensitive carrier.
Mitochondria were incubated as described in the legend of Fig. 2 with 2 mM ATP and 1 µM atractylate present in all samples with varying
concentrations of [3H]GMP. The data were obtained and
presented as described for top. The symbols represent the
average of the velocity of uptake of GMP for 2 experiments. The
best-fit line to the data points for the carrier-mediated uptake curve
was calculated by computer using the equation V = Vmax[GMP]/(Km + [GMP]) (SigmaPlot 5.0). A good fit was obtained between 0.05 and 25 mM GMP with Vmax = 36.8 ± 1.4 pmol · mg 1 · min 1,
Km = 4.1 ± 0.4 mM, and
r2 = 0.996. The predicted line
underestimates the rates below 0.05 mM.
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The atractyloside-insensitive uptake of guanine nucleotide was
more complex, having the ability to transport all three phosphorylated forms of guanine nucleotide. This uptake process could represent a
single carrier capable of transporting all three forms of guanine nucleotide with similar kinetics. Alternatively, the process could represent more than one carrier, each with specificity for a single nucleotide form. We chose to evaluate the kinetic constants of this
carrier by first using [3H]GTP. This was accomplished by
measuring the velocity of uptake of label as a function of
[3H]GTP medium concentration in the presence of
atractyloside and 2 mM ATP (Fig. 7, middle). The initial
velocity was determined by uptake over the first 15 min of incubation.
As above, these data were fit to the carrier-mediated transport
equation by computer, yielding a curve that fit the data well between
0.025 and 25 mM GTP. This curve described the presence of a potential
carrier with a Vmax = 48.7 ± 1.4 pmol
GTP · mg protein
1 · min
1
and a Km = 4.4 ± 0.3 mM (Fig. 7,
middle). Below 0.01 mM GTP, the measured rate of uptake
significantly exceeded the rate predicted from this curve. This
discrepancy may be attributed to another carrier that has a very low
Vmax (<0.1 pmol
GTP · mg
1 · min
1) and a
high-affinity Km (~0.01 mM). HPLC analysis of
the matrix guanine nucleotide pools under these conditions showed that
the vast majority of the label remained in guanine nucleotides
(8), making it unlikely that the faster uptake at low GTP
concentrations was caused by uptake of a small amount of contaminating
radioactivity in the commercially prepared product. In any event, at
physiological levels of guanine nucleotide [0.42 mM
(8)], this discrepancy was inconsequential. As noted in
Fig. 1A, when label was added as [3H]GTP,
~30% of the label was fairly quickly converted to
[3H]GDP; thus uptake probably represented transport of
both species of nucleotide. To further evaluate
atractyloside-insensitive transport, the uptake of label as a function
of [3H]GMP concentration in the medium was determined
(Fig. 7, bottom). As noted earlier, under this condition,
the label remained predominantly as [3H]GMP (Fig.
1C). The results were very similar to the results observed
for [3H]GTP addition. The best-fit curve of
[3H]GMP uptake fit the data well between 0.05 and 25 mM
GMP and described the presence of a potential carrier with a
Vmax = 36.8 ± 1.4 pmol
GMP · mg
1 · min
1 and a
Km = 4.1 ± 0.4 mM (Fig. 7,
bottom). As was noted for [3H]GTP addition,
below 0.05 mM GMP, the measured rate of uptake significantly exceeded
the rate predicted from this curve and suggested another carrier with
low Vmax (~1 pmol
GMP · mg
1 · min
1) and
high-affinity Km (~0.05 mM).
 |
DISCUSSION |
From the results presented in this paper, we propose that guanine
nucleotides can be transported into the matrix of isolated heart
mitochondria by two mechanisms. The first mechanism is carrier mediated, with a Vmax of 946 ± 53 pmol · mg
1 · min
1 and a
Km of 2.9 ± 0.3 mM for GDP. This carrier
is atractyloside sensitive, temperature dependent, capable of active
transport, prefers GDP to GTP, does not transport GMP or IMP, and only
transports GDP when adenine nucleotide levels are limiting (Fig. 4).
This carrier is likely to represent the ATP/ADP translocase, a major protein of the inner membrane that catalyzes the electrogenic exchange
of ADP and ATP across the membrane (for review, see Ref. 6). In active mitochondria, the net activity of this
carrier is to promote the rapid (ms) exchange of medium ADP for matrix ATP. The ADP/ATP translocase is encoded by a family of genes, with the
liver and heart expressing different forms of the protein (6). In mitochondria isolated from the heart, but not the
liver, this carrier has been reported to catalyze
atractyloside-sensitive transport of adenine nucleotide from the
mitochondria to the medium in exchange for phosphate and thus may be
capable of net transport (11). Transport of guanine
nucleotides on the ATP/ADP translocase has not been previously reported
and was only detected in this study by measuring uptake of labeled
guanine nucleotide when adenine nucleotide was omitted from the medium.
The Vmax of GDP uptake on this carrier is
several orders of magnitude below the Vmax of
ATP/ADP exchange. Because the heart expresses a specific form of the
translocase, it is not possible to extrapolate this result to other
tissues. In any event, under physiological concentrations of adenine
and guanine nucleotide, our results suggest that the translocase may
not be involved in guanine nucleotide transport in vivo.
The second mechanism of guanine nucleotide transport is consistent with
a carrier-mediated process that is atractyloside-insensitive, temperature dependent, and appears to transport all three
phosphorylated forms of guanine nucleotide equally. However, the
maximum velocity of this carrier(s) is low, 48.7 pmol · mg
1 · min
1 with a
Km of 4.4 mM for GTP/GDP and 36.8 pmol · mg
1 · min
1 with a
Km of 4.1 mM for GMP, far lower than any other
known carrier. This may suggest a limited number of carriers in the
membrane or a very slow rate of transport after binding nucleotide.
Nevertheless, we believe we have demonstrated that this is a bona fide
physiological mechanism of uptake of guanine nucleotide and is not
caused by potential artifacts of the system. The
atractyloside-insensitive uptake is unlikely to be caused by residual
uptake via incomplete inhibition of the ATP/ADP translocase because the
amount of atractylate used (1 µM) maximally inhibited labeled ATP
uptake (Bentley and McKee, personal observation) and because increasing
the concentration of atractylate had no further effect on labeled GTP
uptake. Furthermore, as shown in Fig. 4, the addition of increasing
amounts of ATP maximally reduced the uptake of labeled GTP to the same
level as that observed in the presence of 1 µM atractylate, again
suggesting that the remaining uptake of GTP is not via the adenine
nucleotide translocase. The temperature dependence of uptake together
with the failure to transport closely related molecules of IMP,
guanine, and guanosine clearly indicate that uptake is not caused by a small number of damaged mitochondria or the transient opening of a pore
or channel, since neither of these processes would be able to provide
the specificity of transport noted. It is also unlikely that uptake is
caused by contaminants in the radioisotope, since such uptake should be
dependent only on the amount of isotope added and should not be
affected by addition of various levels of unlabeled guanine nucleotide.
Our data throughout these experiments show a strong and consistent
correlation between the concentration and specific radioactivity of
guanine nucleotide and the rate of uptake of label that cannot be
explained by label contamination. Furthermore, HPLC analysis of matrix
contents in the presence of atractyloside demonstrated that the vast
majority of the label in the matrix was present in guanine nucleotides
(8). Lastly, although guanine nucleotide uptake is slow,
the process supports biogenesis, not metabolism, and mitochondria in
postmitotic tissue do not require a more rapid system. The only
reaction that consumes GTP in the matrix is RNA synthesis, and, under
steady-state conditions of RNA synthesis and degradation, this may be
replenished by GMP, provided that the matrix contains an enzyme capable
of phosphorylating GMP. We demonstrated in this study that isolated
heart mitochondria (Fig. 1) contain a low-activity enzyme capable of
this phosphorylation; however, whether this enzyme resides in the
intermembrane space, in the matrix, or both, is not yet known.
Mitochondria, like all cellular components, are degraded and turn over
even in nondividing tissues and thus must be capable of continuing
growth and division. It is this process that requires the net uptake of
guanine nucleotide. At an intracellular guanine nucleotide
concentration of 0.42 mM (8), this carrier would be
capable of uptake at a rate of 255 pmol · mg
protein
1 · h
1. This rate is
sufficient to produce the endogenous matrix concentration of guanine
nucleotide (0.96 mM; Table 1 in Ref. 8) in the 1 µl
space of 1 mg of mitochondrial protein in <4 h. This would seem to be
more than adequate to account for mitochondrial turnover. In rapidly
growing tissues, it is possible that the number of guanine nucleotide
carriers in the inner membrane may be increased.
Because the results in this paper only address the influx of guanine
nucleotide from the medium to the matrix, it is not yet possible to
conclude whether this carrier is a simple uniport carrier or a more
complex symport or antiport carrier that depends on a second molecule
for transport. The transport system most similar to the
atractyloside-insensitive process described here is the MgATP/phosphate
exchange carrier, which has been characterized in liver and kidney
mitochondria and is insensitive to atractyloside and bongkrekic acid
(1, 2). This adenine nucleotide carrier is thought to be
important in the net transport of adenine nucleotide across the inner
membrane. An analogous role is proposed here for guanine nucleotide
transport. However, significant differences exist between these two
carrier systems. Mersalyl and N-ethylmaleimide, reagents
that attack active sulfhydryl residues, have been reported to inhibit
the MgATP/phosphate exchange carrier (2) but had no effect
on guanine nucleotide uptake. The MgATP/phosphate exchange carrier is
also reported to have rates (in liver) an order of magnitude above
those noted here for the guanine nucleotide carrier. The increased rate
may reflect the increased concentration and role of adenine nucleotide
in the mitochondria.
To our knowledge this is the first report of a transport process that
accounts for the movement of guanine nucleotides from the cytoplasm
into the mitochondrial matrix to support mitochondrial biogenesis. In
recent work, we have found that a similar transport process may
function in moving deoxythymidine nucleotides from the cytoplasm into
the matrix in support of mitochondrial biogenesis (McKee and Bentley,
unpublished observations).
This work was supported by a Grant-in-Aid from the American Heart
Association, National Chapter.
Address for reprint requests and other correspondence: E. E. McKee, South Bend Center for Medical Education, B15 Haggar Hall, Univ. of Notre Dame, Notre Dame, IN 46556 (E-mail:
mcKee.6{at}nd.edu).
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.