cell biochemistry and function
Cell Biochem Funct (2015)
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/cbf.3089
AMP promotes oxygen consumption and ATP synthesis in heart
mitochondria through the adenylate kinase reaction: an NMR
spectroscopy and polarography study
Nicolai M. Doliba1, Andriy M. Babsky1,2*, Nataliya M. Doliba3, Suzanne L. Wehrli4 and Mary D. Osbakken5
1
University of Pennsylvania, Philadelphia, PA, USA
Ivan Franko National University of Lviv, Lviv, Ukraine
3
Actelion Clinical Research, Cherry Hill, NJ, USA
4
Children’s Hospital of Philadelphia, Philadelphia, PA, USA
5
Osbakken Consulting, LLC, and Visiting Research Professor, School of Biomedical Engineering, Science and Health Systems, Drexel
University, Philadelphia, PA, USA
2
Adenylate kinase plays an important role in cellular energy homeostasis by catalysing the interconversion of adenine nucleotides. The goal of
present study was to evaluate the contribution of the adenylate kinase reaction to oxidative ATP synthesis by direct measurements of ATP
using 31P NMR spectroscopy. Results show that AMP can stimulate ATP synthesis in the presence or absence of ADP. In particular, addition
of 1 mM AMP to the 0.6 mM ADP superfusion system of isolated superfused mitochondria (contained and maintained in agarose beads) led
to a 25% increase in ATP synthesis as measured by the increase in βATP signal. More importantly, we show that AMP can support ATP
synthesis in the absence of ADP, demonstrated as follows. Superfusion of mitochondria without ADP led to the disappearance of ATP γ,
α and β signals and the increase of Pi. Addition of AMP to the medium restored the production of ATP, as demonstrated by the reappearance
of γ, α and β ATP signals, in conjunction with a decrease in Pi, which is being used for ATP synthesis. Polarographic studies showed Mg2+
dependence of this process, confirming the specificity of the adenylate kinase reaction. Furthermore, data obtained from this study demonstrate, for the first time, that different aspects of the adenylate kinase reaction can be evaluated with 31P NMR spectroscopy. Copyright ©
2015 John Wiley & Sons, Ltd.
SIGNIFICANCE OF RESEARCH PARAGRAPH
The data generated in the present study indicate that 31P NMR spectroscopy can effectively be used to study the adenylate kinase reaction
under a variety of conditions. This is important because understanding of adenylate kinase function and/or malfunction is essential to understanding its role in health and disease. The data obtained with 31P NMR were confirmed by polarographic studies, which further strengthens
the robustness of the NMR findings. In summary, 31P NMR spectroscopy provides a sensitive tool to study adenylate kinase activity in different physiological and pathophysiological conditions, including but not exclusive of, cancer, ischemic injury, hemolytic anemia and neurological problems such as sensorineural deafness.
key words—adenylate kinase;
31
P NMR; oxygen consumption; AMP; ATP synthesis; heart mitochondria
INTRODUCTION
Enzyme systems that produce ADP may be capable of stimulating respiration and ATP synthesis. Hexokinase, adenylate kinase and mitochondrial creatine kinase all catalyse
reactions that produce ADP and thus may play a role in
cellular nucleotide metabolism or control of mitochondrial
oxidative phosphorylation.1,2 Adenylate kinase (also known
as myokinase) is a phosphotransferase enzyme that catalyses
*Correspondence to: Andriy Babsky, Department of Physiology of Human
and Animals, Ivan Franko National University of Lviv, 4 Hrushevsky St.,
Lviv, Ukraine 79005.
E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
the conversion of two moles of ADP to ATP and AMP, thus
equilibrating ADP with ATP and AMP (2ADP⇔ATP
+ AMP). In muscle, the concentration of ATP is typically
7–10 times higher than ADP and 100 times higher than
AMP. The rate of oxidative phosphorylation is controlled
by the availability of ADP. Adenylate kinase facilitates
transfer and utilization of γ and β phosphates in the ATP
molecule.3,4 In skeletal muscles, ATP levels decrease by
no more than 20% during intensive exercise. At the same
time, ADP levels may increase by 50%, and AMP levels,
which are in the micromolar range, may increase by
300%.5 AMP activates several metabolic pathways, including glycolysis, glycogenolysis and fatty acid oxidation (particularly in muscle tissues), to ensure that ATP homeostasis
Received 11 August 2014
Revised 3 December 2014
Accepted 9 December 2014
n. m. doliba et al.
is maintained.6 Thus, mitochondria maintain high levels of
ATP because of both adenylate kinase and oxidative phosphorylation. In contrast, adenylate kinase activates ADP
production from ATP and AMP when the myocyte’s need
for ATP is low.
It is known that adenylate kinases may be found in cytoplasm (AK1) or in mitochondria (AK2, AK3,
AKL3L1).7 Furthermore, it has been shown that the enzymatic isoform, AK3L1, is specifically associated with mitochondrial matrix,8 whilst AK2 is found in the
mitochondrial intermembrane space.9,10 Knock out of the
human gene encoding proteins for adenylate kinase
(AK1) desynchronized inorganic phosphate turnover at
ATP-consuming sites and ATP synthesis sites. This decreases energetic signal processes in the post-ischemic
heart resulting in insufficient coronary reflow following
ischemia-reperfusion injury.7
Krishnamurthy et al. show that adenylate kinase is associated with Mg2+, diadenosinepentaphosphate and four coordinated water molecules.4 Mg2+, coordination water and
surrounding charged residues uphold the geometry and distances of the AMP α-phosphate and ATP β and γ phosphates. This is sufficient to support an associative reaction
mechanism for phosphate transfer.4
Along these lines, it has been shown that AMP has similar
effects as ADP on respiration of mitochondrial suspensions
under aerobic conditions.11,12 The authors hypothesized that
AMP reacts with endogenous ATP by the action of adenylate kinase to produce ADP, which then activates oxidative
phosphorylation.
There are several enzymatic methods that can be used to
analyse adenylate kinase activity at one point in time, or at
different times by continuous sampling from an enzyme reaction mixture. These techniques generally use purified enzymes.9,10,8 In the present study, an NMR spectroscopy
method was developed that allows continuous measurement of adenylate kinase activity in intact organelles in real
time. In this regard, we used 31P NMR spectroscopy in
conjunction with superfused mitochondria incorporated
into agarose beads.13 This approach allows excellent mitochondrial metabolic stability and provides a unique tool for
investigation of many aspects of intact mitochondrial
function in real time.
MATERIALS AND METHODS
Preparation of isolated mitochondria
Heart mitochondria were isolated from 3–4 rat hearts (for
each NMR experiment) or from 1–2 hearts (for each polarographic experiment) using differential centrifugation according to a generally accepted scheme, with modifications
which provide protection of mitochondrial native state.14,15
To ensure optimal protection of the mitochondrial native
state, rat hearts were quickly excised under Nembutal anesthesia (50 mg/kg, injected intraperitoneally), exsanguinated,
chilled and weighed. Then, hearts were homogenized in
buffer containing: 300 mM sucrose, 10 mM HEPES
Copyright © 2015 John Wiley & Sons, Ltd.
(pH = 7.4) and 1 mM EDTA. To stabilize the mitochondrial
membrane, 0.25% BSA was added to the homogenizing
media. An Akhmerov’s homogenizer, with a motor-driven
Teflon pestle containing a metal core in the middle of the
pestle, was used.16 This allowed increase in the quality of
homogenization and decrease in homogenization time. After
initial centrifugation (7 min at 480 g), the pellet-containing
nuclei and other cellular debris were discarded. The supernatant was centrifuged at 10 000 g for another 15 min to
sediment the mitochondrial fraction.
To document the physiological state of isolated mitochondria, the respiration control index (RCI) of a sample
from each mitochondrial preparation was determined. Respiration rates were measured using pyruvate plus malate as
substrate. State 3 respiration was determined by addition
of 320 nmol ADP to the mitochondria in the solution listed
in the preceding text. State 4 respiration was defined as oxygen usage after ADP phosphorylation. The RCI was determined as the ratio of State 3/State 4 respiration rates. The
RCI of the isolated mitochondrial preparations was generally high and in the range of normal RCIs from 5 to 6. Mitochondrial preparations with a RCI less than 5.5 were not
used. Furthermore, the rates of oxygen consumption before
and after ADP phosphorylation were almost equal, indirectly indicating full recovery of NAD, restoration of the
proton gradient and native state of inner mitochondrial
membranes.
Polarographic experiments
Respiration assays were performed using a Gilson oxygraph
equipped with a Clark electrode. Reactions were conducted
at 28 °C in a 1.5-ml chamber containing 2–3 mg of the mitochondrial fraction suspended in the respiration buffer of
220 mM sucrose, 50 mM KCl, 1 mM KH2PO4 and 5 mM
HEPES (pH = 7.4). Documentation of the mitochondrial
physiological state was carried out as described earlier under
‘Preparation of isolated mitochondria’.
NMR experiments preparation
Prior to all NMR experiments, mitochondrial physiological
state was checked by measuring the RCI of a mitochondrial
aliquot, as described above. Mitochondria were placed in
agarose beads (bead size = 800–1000 μm, 2000–2500
beads/cm3)13,17 to maintain them in a stable environment
and prevent them from escaping from the superfusion system. Approximately 7–10 mg of mitochondrial protein, of
the initial 30 mg that was isolated, was incorporated into
the beads. Mitochondria were suspended in 1 ml of buffer,
which contained 220 mM sucrose, 50 mM KCl, 1 mM
KH2PO4, 2 mM MgCl2, 0.5 mM EDTA, 20 mM HEPES
(pH 7.4), 0.6 mM ADP and 2 mM ATP, 2.5 mM creatine,
5 mM pyruvate plus 5 mM malate. Mitochondria, suspended
in this buffer, were then added to 1 ml of 6% agarose (Sigma
type VII, low gelling temperature) and mixed at 37 °C. This
suspension was then decanted into 50 ml of paraffin oil (37 °
C) and stirred continuously. The interaction (difference in
surface tension) of the oil and agarose caused bead
Cell Biochem Funct (2015)
role of amp in mitochondrial atp synthesis
formation. Beads were cooled to 10 °C by adding ice to the
water bath while stirring continuously for 5 min to allow
them to become firm and to maintain their shape. Beads
containing mitochondria were suspended in buffer without
ATP, and washed several times. The initial use of ATP in
the medium was to maintain mitochondrial stability until
they could be superfused in beads.
Superfusion apparatus
Beads containing mitochondria were placed in a 10 mm
diameter glass NMR tube and maintained in place by a
filter (100 μm pore size). Each NMR sample contained
approximately 1.5 ml of the beads suspended in 1 ml
buffer (7–10 mg mitochondrial protein or 84–120 μl of
mitochondria by volume). The mitochondria and the capillary
tube containing a methylene diphosphonate standard were
placed within the sensitive volume of the NMR coil. The
superfusion medium containing ADP, but without ATP, was
equilibrated with 95% O2, 5% CO2 at 28 °C and was introduced through an in-flow line (at 2.7 ml/min). A suction line,
placed above the beads, removed the superfusate.
The superfusate was not recirculated. The superfusion
medium used for the ischemia-reperfusion studies contained
(in mM): sucrose, 150; KCl, 50; KH2PO4, 1; HEPES, 20 or
30; MgSO4, 2; EDTA, 0.5; creatine, 2.5; α-ketoglutarate
(KG), 5; and ADP, 0.6.
31
P NMR method
The 31P NMR measurements were performed with a Bruker
AM-400 spectrometer at 162 MHz. 31P NMR spectra were
acquired (without proton decoupling) consecutively in
10-min periods (1000 transients) for up to 10 h. The following conditions were used: pulse width 36°, sweep width
13 kHz, 16 K data points, repetition time 0.6 s. Free induction decays were processed with a Lorentz-Gauss window
function for resolution enhancement (Bruker parameters:
line broadening = 8, maximum of the Gaussian
function = 0.003). The spectra were referenced to the
resonance of PCr set at 0 ppm.
Figure 1. Dependence of heart mitochondria ATP, PCr and AMP on external ADP concentration. ATP synthesis was measured by following the
βATP signal. Peak areas for ATP, PCr and AMP were normalized to the
reference of a methylene diphosphonate standard
by monitoring the βATP signal. Mitochondrial ATP production reached saturation at high concentrations of ADP
(~3 mM) (Figure 1). During synthesis of ATP from ADP,
AMP was found to increase as well. In addition, at the
higher concentrations of ADP (1.2–5.0 mM), the PCr signal
disappeared, indicating that PCr also can be used for ATP
synthesis in the creatine kinase reaction.
Stimulation of ATP synthesis by AMP:
31
P NMR data
Figure 2 (Baseline) presents spectra from heart mitochondria
oxidizing 5 mM pyruvate plus 5 mM malate in the presence
of 0.6 mM ADP, 1 mM phosphate, and 2.5 mM creatinine at
28 °C. Each spectrum clearly displays the resonances of
ATP (which is absent from the superfusate), as well as Pi
and βADP. Whilst the α-phosphate signals of ATP and
ADP overlap, their terminal phosphates are clearly separated
(γATP and βADP). Addition of 1 mM of AMP to superfusate
Statistical analysis
31
P NMR data are presented as typical spectra (n = 4–5). All
polarographic data are presented as the mean ± standard
error of mean, and represent the range across a cohort of animals. Analysis of the polarographic data was performed
using the Student’s t-test. A p-value < 0.05 was used to
define statistical significance.
RESULTS
31
Regulation of mitochondrial ATP synthesis by ADP
31
P NMR spectroscopy methods are ideally suited to study
the regulation of ATP synthesis by various substrates and
regulators. In these studies, we evaluated the effects of increasing ADP concentration on ATP synthesis, as measured
Copyright © 2015 John Wiley & Sons, Ltd.
Figure 2. Effect of addition of AMP on P NMR spectra of superfused
heart mitochondria. Heart mitochondria were superfused with oxygenated
31
buffer at 28 °C. P NMR spectra were acquired consecutively (without proton decoupling) in 10-min periods (1000 transients). Peak assignments are
indicated on the figure: MDP = methylene diphosphonate standard;
Pi = inorganic phosphate; PCr = phosphocreatine; ADP = adenosine diphosphate (α, β); ATP = adenosine triphosphate (γ, α, β)
Cell Biochem Funct (2015)
n. m. doliba et al.
31
31
Figure 3.
P NMR spectra of superfused heart mitochondria with ADP
(Baseline), without ADP and AMP, and with AMP but without ADP. Refer
to Figure 2 for definitions and explanation of other experimental conditions
containing 0.6 mM of ADP leads to a 25% increase in ATP
synthesis as measured by following the βATP signal.
The next set of experiments showed that AMP can support ATP synthesis in the absence of ADP. Superfusion of
mitochondria without ADP and AMP leads to the disappearance of the ATP signals [Figure 3 (middle spectrum)]. Addition of AMP (1 mM) to the medium restored the production
of ATP, which could be followed by the appearance of the γ,
α and β ATP signals (upper spectrum). The Pi signal decreased, as it was being used for ATP synthesis. These results indicate that AMP can support ATP synthesis in the
absence of exogenous ADP.
AMP-supported post-ischemic recovery
AMP may play an important role in heart muscle during
post-ischemic recovery. Figure 4 presents 31P spectra from
superfused heart mitochondria before, during and after ischemia. The ischemia was initiated by 20-min stop flow,
followed by 30-min re-superfusion. Ischemia led to significant increase in Pi peak, to the complete disappearance of
all ADP and ATP peaks whilst AMP peak became clearly
visible at ppm 6.0 (middle spectrum). The full recovery of
ADP and ATP peaks during 30-min re-superfusion was accompanied by the disappearance of the AMP peak (upper
spectra). This finding suggests that AMP could be an important substrate for direct or indirect (through ADP) ATP
synthesis.
AMP-stimulated oxygen consumption
Figure 5 presents the oxygen electrode tracing of heart mitochondria oxidizing 1 mM pyruvate plus 1 mM malate.
Addition of 0.1 mM AMP greatly stimulates oxygen consumption in Mg2+-containing buffer (curve 1) but has no
Copyright © 2015 John Wiley & Sons, Ltd.
Figure 4. Effect of ischemia on P NMR spectra of superfused heart mitochondria. Ischemia: 20-min stop-flow superfusion; recovery: 30-min resuperfusion. The superfusion medium used for the ischemia-reperfusion
studies contained (in mM): sucrose, 150; KCl, 50; KH2PO4, 1; HEPES,
20 or 30; MgSO4, 2; EDTA, 0.5; creatine, 2.5; α-Ketoglutarate, 5; ADP,
0.6. Refer to Figure 2 for definitions and explanation of other experimental
conditions
effect in Mg2+-free buffer (curve 2). Addition of Mg2+ to the
medium containing 0.1 mM AMP (curve 2) increased the
respiration to the same level as seen in curve 1. An inhibitor
of oxidative ATP synthesis, oligomycin, completely
abolished the effect of AMP (curve 3). Intensive activation
of respiration by FCCP indirectly shows a strong coupling
between respiration and oxidative phosphorylation in both
curves 1 and 3. Statistical analysis of oxygen consumption
data is summarized in Table 1.
To evaluate the Km for AMP, heart mitochondria
oxygen uptake was activated by different concentrations
of AMP. Figure 6 represents the dependence of the rate
of mitochondrial respiration on AMP concentrations in
the polarographic chamber (in mM): 0.025, 0.05, 0.1,
0.2, 0.3 and 0.6. The Km for AMP was found to be 20.7
± 3.50 μM.
DISCUSSION
In this study, the contribution of the adenylate kinase reaction to oxidative ATP synthesis in rat heart mitochondria
was estimated using both 31P NMR spectroscopy and polarographic oxygen consumption measurements. In isolated mitochondria superfused with 0.6 mM of ADP, the
addition of 1 mM AMP led to an increase in ATP, as measured by the increase of the βATP signal. The data demonstrate that AMP can support ATP synthesis. Superfusion of
mitochondria without ADP led to disappearance of ATP
signals and increase in Pi. Addition of AMP to the medium
restored the production of ATP, which was followed by the
Cell Biochem Funct (2015)
role of amp in mitochondrial atp synthesis
Figure 5. Effect of AMP on oxygen uptake of heart mitochondria. Respiration was measured polarographically in 1.5 ml of incubation mixture (see
Materials and Methods); temperature 28 °C. The mitochondria were added
at a concentration of 1.28 mg of protein per 1.5 ml. 1 and 3 – with MgCl2,
2 – w/o MgCl2. Concentrations: MgCl2, 1 mM; AMP, 0.1 mM; oligomycin
(Oligo), 2.5 μM. Substrates: pyruvate, 1 mM plus malate, 1 mM. The numbers next to each curve indicate the rate of respiration (ng-atoms O per min
per mg protein). n = 8 (curve 1), 3–4 (curves 2 and 3)
appearance of the γ, α and β ATP signals. The Pi was decreased as it was being used for oxidative phosphorylation
of ADP.
The data demonstrate that AMP, as well as ADP, stimulates heart mitochondrial oxygen consumption. The Km for
AMP was found to be 21 μM. It has been shown that the mitochondrial adenylate kinase (AK2 isoform) in heart muscle
has the highest affinity (lowest Km) for AMP (≤10 μM)
amongst AMP metabolizing enzymes.7,18 In addition, in
the mitochondria-like kinetoplast organelle, the Km for
AMP (AK2) is 74 μM.19 Thus, we believe that the current
data (21 μM ) obtained with polarography are close enough
to be in a comparable range with other studies when different techniques were used.
Mitochondrial ATP synthesis depends on the amount of
ADP available. A dose dependency curve for ATP production shows that ATP synthesis reaches saturation at high
Table 1.
concentrations of ADP (Figure 1). These results are different
from literature data on mitochondrial oxygen consumption,20 which show saturation of ATP synthesis at much
lower concentrations of ADP with the Km 20–30 μM. The
saturation behaviour observed in the current experiments
may represent not only oxidative ATP synthesis but also
other rate limiting steps, such as transport processes or
the combination of the simultaneous function of several
metabolic pathways. For example, ATP produced by the
myokinase reaction may contribute to the ATP produced
by oxidative processes, thus allowing for higher levels of
ATP production. The evidence supporting this hypothesis
is that the AMP level increased as the exogenous ADP
concentration was increased. The level of PCr, in contrast,
decreased and the signal disappeared at higher concentrations of ADP (1.2 mM of ADP), indicating that PCr
also can be used for ATP synthesis in the creatine kinase
reaction.
The current data show that NMR spectroscopy can be
used to study adenylate kinase activity, which plays an important role in cellular energy homeostasis in normal, as
well as in pathological conditions, such as hypoxia and ischemia. NMR spectroscopy allows evaluation of the adenylate kinase reaction by measuring changes in the 31P ATP
signals. The polarographic studies supported the findings
of the 31P NMR data; i.e., that ATP synthesis was maintained by AMP via Mg2+-related adenylate kinase reaction
because it is strongly dependent on the presence of Mg2+.
This study demonstrates that 31P NMR is an excellent tool
to evaluate adenylate kinase activity under different conditions that play a role in pathophysiology related to changes
in this enzyme system. It has been shown that there is a connection between adenylate kinases malfunction and progression of cancer,9 ischemia-induced injury,7 hemolytic
anemia21 and sensorineural deafness.22
In summary, these data demonstrate the importance of the
adenylate kinase reaction to support oxidative ATP synthesis as measured by 31P NMR spectroscopy. This technology
can be effectively used to evaluate simultaneous interaction
of intra-mitochondrial processes. In addition, the present
data show that AMP can serve not only as a regulator but
also as an indirect substrate for oxidative phosphorylation
by reacting with endogenous ATP to produce ADP, which
then activates oxidative phosphorylation.
Rates of oxygen consumption under experimental conditions for investigation of AMP stimulated respiration in isolated heart mitochondria
Experimental conditions
Before AMP
With AMP
After AMP
+MgCl2
n
MgCl2
+MgCl2
n
+MgCl2 and oligomycin,
n
24.8 ± 1.49
8
21.1 ± 5.69
158 ± 12.9*
8
26.2 ± 6.33
159 ± 38.3*
4
26.2 ± 1.89
4
130 ± 12.6*
8
—
261 ± 8.53*
8
—
—
231 ± 14.9*
4
4
22.6 ± 1.64
4
With FCCP
№ of Curves (see Figure 5)
1
2
3
Note. mean ± standard error of mean. Respiration rate values are presented in ng-atoms O/min × mg protein.
*p < 0.05 (vs. Before AMP). Substrates: pyruvate, 1 mM plus malate, 1 mM. Concentrations: MgCl2, 1 mM; AMP, 0.1 mM; oligomycin, 2.5 μM.
Copyright © 2015 John Wiley & Sons, Ltd.
Cell Biochem Funct (2015)
n. m. doliba et al.
5.
6.
7.
8.
9.
10.
11.
Figure 6. Relationship between the AMP-stimulated respiration and the
AMP concentration in the medium. Data represent the dependence of the
rate of mitochondrial respiration on AMP concentrations in the polarographic chamber (in mM): 0.025, 0.05, 0.1, 0.2, 0.3 and 0.6. The Km for
AMP was found to be 20.7 ± 3.50 μM. Refer to Figure 5 for explanation
of other experimental conditions
13.
CONFLICT OF INTEREST
14.
12.
The authors have declared that there is no conflict of
interest.
15.
ACKNOWLEDGEMENTS
This paper is dedicated to the memory of Dr. Stepan K.
Hordiy (1936–2010), teacher, colleague and friend, who always generously inspired us to study the role of adenylate
kinase in energetic processes.
16.
17.
18.
19.
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