In this article, it is shown that, under physiological conditions, ADP may be formed in mammalian tissues by the disproportionation of AMP, consistent with an AMP–AMP phosphotransferase
Trang 1nucleotide metabolism
Daniela Vannoni1,*, Roberto Leoncini1,*, Stefania Giglioni1, Neri Niccolai2, Ottavia Spiga2, Emilia Aceto1and Enrico Marinello1
1 Department of Internal Medicine, Endocrine-Metabolic Sciences and Biochemistry, University of Siena, Italy
2 Department of Molecular Biology, University of Siena, Italy
Purine nucleotides are precursors of nucleic acids and
participate in many metabolic pathways as substrates,
coenzymes and energy sources Much attention has
been focused on the roles and intracellular levels of
AMP, ADP and ATP in various tissues under normal
and pathological conditions [1–3] Their relationships
to genetic diseases, blood disorders, drugs, tumours
and other pathologies have been studied extensively
[4–6]
Although ATP formation occurs via several
well-known mechanisms, ADP is thought to be formed
only from ATP, either by the adenylate kinase reaction
or by ATPase-mediated hydrolysis The possibility that
ADP can be formed by de novo synthesis from
low-energy precursors has never been investigated fully Even less studied is the possibility that ADP might be synthesized from low-energy precursors under special conditions, such as ischaemia and hypoxia, in which massive depletion of ATP and elevation of AMP are known to occur [7–10]
In this article, it is shown that, under physiological conditions, ADP may be formed in mammalian tissues
by the disproportionation of AMP, consistent with an AMP–AMP phosphotransferase reaction This reaction
is carried out by enzymes of purine metabolism which, under specific cellular conditions, associate in a biolog-ical network and cooperate in a reaction not reported previously
Keywords
adenosine deaminase; adenosine kinase;
adenylate kinase; ADP; ATP
Correspondence
R Leoncini, Department of Internal
Medicine, Endocrine-Metabolic Sciences
and Biochemistry, Via A Moro 2,
53100 Siena, Italy
Fax: +39 0577 234285
Tel: +39 0577 234287
E-mail: leoncini@unisi.it
*These authors contributed equally to this
work
(Received 16 July 2008, revised 24
September 2008, accepted 5 November
2008)
doi:10.1111/j.1742-4658.2008.06779.x
Crude rat liver extract showed AMP–AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adeny-late kinase, also known as myokinase (EC 2.7.4.3), and adenosine kinase (EC 2.7.1.20) The activity was duplicated using the same enzymes purified from recombinant sources The reaction requires physical contact between myokinase and adenosine kinase, and the net reaction is aided by the pres-ence of adenosine deaminase (EC 3.5.4.4), which fills the gap in the energy balance of the phosphoryl transfer and shifts the equilibrium towards ADP and inosine synthesis The proposed mechanism involves the association of adenosine kinase and myokinase through non-covalent, transient interac-tions that induce slight conformational changes in the active site of myokinase, bringing two already bound molecules of AMP together for phosphoryl transfer to form ADP The proposed mechanism suggests a physiological role for the enzymes and for the AMP–AMP phosphotrans-ferase reaction under conditions of extreme energy drain (such as hypoxia
or temporary anoxia, as in cancer tissues) when the enzymes cannot display their conventional activity because of substrate deficiency
Abbreviations
ADA, adenosine deaminase; AdK, adenosine kinase; CE, capillary electrophoresis; MD, molecular dynamics; MK, myokinase.
Trang 2These enzymes are well known for their
conven-tional reactions:
(a) Adenylate kinase, also called myokinase (MK),
which catalyses the reversible reaction:
AMPþ ATP $ ADP (b) Adenosine kinase (AdK), which catalyses the
irreversible reaction:
Adenosineþ ATP ! AMP þ ADP
(c) Adenosine deaminase (ADA), which catalyses the
irreversible reaction:
Adenosineþ H2O! inosine þ NH3
The AMP–AMP phosphotransferase reaction is:
AMPþ AMP ! ADP þ inosine þ NH3
ADP and inosine were identified by various
meth-ods, including HPLC and diode array analysis; the
enzymes responsible for the reaction were identified
through a complex purification procedure Our
find-ings demonstrate that MK and AdK carried out the
reaction, and ADA enhanced the rate ATP was
formed only in the presence of ADA, and at longer
incubation times, when the ADP concentration passed
the threshold necessary to allow the initiation of the
MK reaction and the AMP concentration decreased
below the inhibitory concentration for MK In this
study, we investigated the complex mechanisms
under-lying this reaction and its physiological role in cell
metabolism
Results
AMP–AMP phosphotransferase reaction
Dialysed rat liver supernatant was incubated with
AMP and Mg2+; HPLC analysis revealed the
forma-tion of two products with retenforma-tion times of 1.3 and
7.1 min, which were identified as inosine and ADP,
respectively (Fig 1) Using purified [32P]AMP as a
sub-strate, ADP formation was consistent with the transfer
of 32P between two molecules of [32P]AMP to form
[32P]ADP[a,bP] Thus, starting from [32P]AMP with a
specific radioactivity of 50 500 ± 1517 d.p.m.Ænmol)1
(mean of five experiments), we obtained ADP with a
specific radioactivity of 108 125 ± 4325 d.p.m.Ænmol)1,
double that of the starting AMP Hydrolysis of the
product yielded two moles of32Piper mole of ADP
These findings indicate that supernatants starting
from low-energy precursors catalyse the AMP–AMP
phosphotransferase reaction:
AMPþ AMP ! ADP þ inosine þ NH3 ð1Þ resulting from reactions (2) and (3):
AMPþ AMP ! ADP þ adenosine ð2Þ Adenosineþ H2O! inosine þ NH3ðADA reactionÞ ð3Þ ADA converts adenosine to inosine and ammonia in stoichiometric amounts with respect to ADP
Enzyme purification and identification by mass spectrometry
Protein purification demonstrated that ADP forma-tion occurred via the activities of MK and AdK, with the cooperation of ADA The crude supernatant and P90d fraction showed AMP–AMP phosphotransferase activity, but any further chromatography led to the loss of activity, presumably because the two different proteins were separated from one another Therefore,
we purified each protein individually by an appro-priate procedure AMP–AMP phosphotransferase activity was restored every time we combined the two separate protein preparations ADA was also isolated When ADA was added to the assay mixture, AMP– AMP phosphotransferase activity was greatly enhanced
The purifications were performed as reported in Table 1 The final SDS-PAGE showed a single band for each protein preparation
Fractions X2 and Y2 were identified as MK and AdK, respectively, by electrospray mass spectrometry The X2 fraction spectra revealed a component with a molecular mass of 26 232.5 ± 0.5 Da (Fig 2) Twenty signals ranging in mass from m⁄ z 780.7 to m ⁄ z 1726.8 were selected and entered into the non-redundant National Center for Biotechnology Information data-base using pro found software The query returned a
Fig 1 Identification of ADP and inosine in the reaction mixture The figure shows the typical HPLC pattern of an assay mixture incubated with crude extract of rat liver (0.5 mg) for 50 min Numbered peaks: 1, inosine; 2, AMP; 3, ADP.
Trang 3significant match with the protein MK (isoenzyme 2)
from Rattus norvegicus The mass signals used in the
search procedure accounted for 72% of the entire MK
sequence, confirming identification The electrospray
mass spectra of Y2 revealed a component with a
molecular mass of 38 344.2 ± 2.1 Da (Fig 3) Twenty
signals ranging in mass from m⁄ z 1235.94 to m ⁄ z
3316.66 were selected, and the query returned a highly
significant match with the protein AdK from R
nor-vegicus The mass signals used in the search procedure
accounted for 80% of the entire AdK sequence,
confirming identification Mass analysis of fractions
X2 and Y2 confirmed the purity of the two purified
proteins (Figs 2 and 3)
AMP–AMP phosphotransferase activity by
recombinant enzymes
One microgram of Escherichia coli-expressed human
recombinant AdK, mixed with commercial
recombi-nant MK and ADA, exhibited AMP–AMP
phospho-transferase activity Commercial preparations of
purified MK and ADA from several sources produced
the same result as the purified rat liver enzymes (AdK
was not commercially available)
General properties of the reaction AMP–AMP phosphotransferase activity was detectable
in different rat tissues (muscle, brain, spleen) and was absolutely specific for AMP The formation of ADP and inosine progressed over time and no products were formed when the protein extract was denatured by heat or acid The time course of the reaction was linear for at least 60 min when only MK and AdK were present When ADA was added, the shape of the curve remained as before for the first 15 min; thereafter, the reaction proceeded linearly for 50 min, at a rate 20 times greater than that for MK and AdK alone (Fig 4)
ADP and inosine formation varied according to the incubation temperature, pH, AMP concentration and
Mg2+ concentration Maximum activity was found at
pH 6.5–6.8, and the activity decreased sharply at pH values above 7.5 and below 5.5 The Km value for AMP was 0.8 mm (Fig 5A) Mg2+ was essential for the AMP–AMP phosphotransferase reaction, with the optimal concentration in the range 0.8–1.5 mm The apparent Km value of Mg2+ was 0.35 mm (Fig 5B) Certain ions added to the incubation mixture inhibited (NHþ
4, Li+ and SO2
4 ) or had no effect (Ca2+ and
Table 1 Purification of rat liver X2 and Y2 proteins The two proteins responsible for AMP–AMP phosphotransferase activity in rat liver were purified The procedure started with a common trunk and had three steps: supernatant production, ammonium sulfate precipitation and dialysis During these steps, the two proteins were not separated and phosphotransferase activity was present in all fractions After DE-52 chromatography, the purification process diverged: non-retained fractions were utilized for the purification of protein X (X1 or X2; finally identified as MK) and part of the retained fractions was used for the purification of protein Y (Y1 or Y2; finally identified as AdK) AMP–AMP phosphotransferase activity was only detected when suitable amounts (0.003–0.5 mg) of fractions from each purification branch were pooled (i.e X1 + Y1, X2 + Y2).Total AMP–AMP phosphotransferase activity is expressed in IU, which corresponds to the number of micromoles of ADP or ADP + ATP formed per minute ND, AMP–AMP phosphotransferase activity was not detectable.
Common trunk
Step
Supernatant
P90d
DE-52
Retained (R)
X protein
X1 Blue Sepharose
X2 Superdex 75
Y protein
Y1 AMP Sepharose
X1 + Y1
Y2 + Y2
Y2 Superdex 75
ND ND ND
ND ND
ND ND ND ND
ND ND
Trang 4PO23 ) on the reaction Zn2+ completely eliminated
phosphotransferase activity
The amounts and ratios of enzymes in the assay
mixture reflected those reported to be present in rat
liver, namely MK@ 0.45 IU and AdK @ 0.015 IU,
with an MK : AdK ratio of approximately 30 [11,12]
In our assay mixture, we used 1–3 lg of pure protein,
corresponding to approximately 0.21–0.63 IU MK and
0.006–0.018 IU AdK, with a ratio of about 10–30
When ADA was present in the assay mixture, ATP
was formed when the incubation time exceeded
15–20 min, and increased with time ATP, with a
retention time of 9.2 min on the HPLC chromatogram,
was identified using the same criteria as for ADP
Using [32P]AMP as a substrate, the ATP formed had a
specific radioactivity of 146 338 ± 7316 d.p.m.Ænmol)1,
three times that of AMP ATP was formed by the
con-ventional MK reaction, when the ADP concentration
in the mixture passed the threshold of 0.05 mm (data
not shown); at lower ADP concentrations, MK activity
was inefficient because of a lack of substrate At longer incubation times, when the ADP concentration exceeded the Kmvalue for MK (0.3 mm) and the AMP concentration fell below the inhibitory concentration (Ki of AMP for MK, 2.13 mm), MK exerted its con-ventional activity, eventually reaching equilibrium [12] The Ki value of AMP for MK was not influenced by the presence of AdK
Reversibility of the reactions
We considered the reversibility of the reactions: AMPþ AMPðAdK;MK;ADAÞ! ADPþ inosine þ NH3 ð1Þ AMPþ AMPðAdK;MKÞ! ADPþ adenosine ð2Þ Reaction (1) was irreversible and reaction (2) also appeared to be irreversible; in the presence of ADP and adenosine alone, MK activity was absolutely
A
B
Fig 2 Electrospray ionization mass spectra of the X2 fraction identified as MK (A) Gaussian-type distribution of multiply charged ions (B) The m ⁄ z spectrum converted to a molecular mass profile by maximum entropy processing The mass profile is dominated by a single com-ponent, showing the purity grade of the protein The molecular mass of the sample was calculated by the processing software associated with the mass spectrometer to a mass accuracy of 0.01%, and can be read directly on the graph.
Trang 5predominant, and so equimolar amounts of AMP and
ATP were formed by the very rapid conventional MK
reaction [see reaction (a) in the introductory section]
Moreover, if we added 0.2 lCi of [14C]adenosine to
the incubation mixture, no [14C]AMP was formed
Reaction mechanism studies – cooperation
between AdK and MK
Micro-equilibrium dialysis experiments
No reaction products were formed when AdK and
MK were separated by a dialysis membrane during the
reaction, regardless of the presence of ADA The
AMP–AMP phosphotransferase reaction could only be
detected when AdK and MK were incubated in the
same chamber, in which case the presence of ADA
only affected the rate of the reaction
Detection of reaction intermediates by HPLC, capillary electrophoresis (CE) and NMR analysis
HPLC, CE and NMR analysis of the incubation mix-ture at several points during the incubation indicated that no intermediate products were formed In all cases,
we observed a decrease in AMP concentration over time and an increase in adenosine or inosine, ADP and ATP concentrations; no other products were detected
Enzyme–phosphate intermediate trapping When enzyme–phosphate intermediate trapping experi-ments were performed, no spot was detected on the autoradiography slide, indicating direct transfer of the phosphoryl group from one AMP molecule to another, without the formation of an intermediate
A
B
Fig 3 Electrospray ionization mass spectra of the Y2 fraction identified as AdK (A) Gaussian-type distribution of multiply charged ions (B) The m ⁄ z spectrum converted to a molecular mass profile using maximum entropy processing The mass profile is dominated by a single component, showing the purity grade of the protein The molecular mass of the sample was calculated by the processing software associ-ated with the mass spectrometer to a mass accuracy of 0.01%, and can be read directly on the graph.
Trang 6phosphoenzyme species When vanadate was added to
the reaction mixture, no inhibition of AMP–AMP
phos-photransferase was observed Moreover, the addition of
[14C]nucleoside (adenosine, guanosine, inosine) did not
promote the formation of [14C]AMP, [14C]GMP or
[14C]IMP The formation of ADP was unchanged
Gel filtration and SDS-PAGE
Assay mixtures incubated for 0 and 60 min were
sub-mitted to gel filtration The resulting chromatograms
showed no differences in retention times, indicating
that no stable protein complex was formed Moreover,
when the same samples were resolved by SDS-PAGE,
no bands were observed at a molecular mass higher
than AdK, ruling out the formation of covalent bonds
between the proteins
Docking simulation studies
We performed a molecular dynamics (MD) simulation
of the interaction between AMP and AdK or MK
using autodock This procedure suggested that, from
an energy point of view, each protein had two binding
sites, either of which could be occupied by AMP gromacs was then used to optimize the molecular models with energy minimizations followed by a 1 ns
MD simulation Using the MD trajectory, possible changes in AMP position and in the network of bonds between the AMP molecules and binding pockets were examined In the case of rat MK, one of the two bound AMP molecules maintained the same location and orientation during all MD runs, whereas the other molecule changed position and approached the first AMP molecule Indeed, the distance between these two molecules was 8.19 A˚ at the beginning of the docking simulation, and 5.61 A˚ at the end of the simulation (Fig 6B), close to the 4.5 A˚ distance between the natu-ral ligands AMP and ATP (Fig 6A) With regard to AdK, the MD simulation showed that the two AMP molecules were bound in positions very distant from each other (Fig 7)
Kinetic analysis Kinetic experiments were performed in the absence
of ADA AMP–AMP phosphotransferase activity
9
A
B
6
3
0
0
200
150
100
50
0
10 20 30
Minutes
Minutes
40 50 60
0 10 20 30 40 50 60
Fig 4 Time course of ADP formation by the AMP–AMP
phospho-transferase reaction (A) 0.38 IU of purified rat liver MK and
0.012 IU of AdK The amount of ADP produced (nmol) is shown on
the ordinate (B) 0.38 IU of purified rat liver MK, 0.012 IU of AdK
and 0.9 IU of ADA The amount of ADP + ATP formed (nmol) is
shown on the ordinate.
Fig 5 (A) Direct and double reciprocal plot of initial velocities with variable AMP concentration (0.0–6.0 m M ) at a constant Mg 2+ concentration (1.0 m M ) (B) Direct plot of the initial velocities with variable Mg 2+ concentration (0.1–1.4 m M ) and constant AMP concentration (4.0 m M ) Values are the mean ± standard deviation
of five experiments Assay mixtures contained 0.15 IU MK, 0.004 IU AdK and 0.3 IU ADA The amount of ADP + ATP formed (nmolÆh) is shown on the ordinate.
Trang 7gradually increased to a plateau, following a typical
hyperbolic curve (Fig 8), and reached a maximum
saturating value when the concentration of AdK
exceeded 2 nmol (AdK : MK ratio > 2000) The Kd
value was 1.496 lm, as determined by the Scatchard
equation [13]
Inhibition experiments
The results of the inhibition assays are presented in
Table 2 Each inhibitor completely blocked the
activity of its respective enzyme, i.e Ap5A inhibited
MK and A134974 inhibited AdK Neither inhibitor
interfered with the progress of the other reaction
AMP–AMP phosphotransferase activity was only
affected by the presence of Ap5A, when it fell to
zero A134974 had only a slight effect on the
AMP–AMP phosphotransferase reaction (< 10%
inhibition)
AMP–AMP phosphotransferase reaction in human colorectal mucosa from cancer patients
Table 3 shows the activities of AdK, MK, ADA and AMP–AMP phosphotransferase in normal and cancer-ous human colorectal mucosa The MK activity did not vary, but the AdK and ADA activities were signifi-cantly elevated (P < 0.0001) in cancer tissue with respect to the surrounding normal mucosa AMP– AMP phosphotransferase activity was only detectable
in tumour tissue
A
B
Fig 6 (A) Backbone superimposition of human (green) and rat
(red) MK The natural ligands (AMP and ATP) and the two AMP
molecules (red) are shown in bold (B) Backbone superimposition of
rat MK before (magenta) and after (green) the MD run Note the
reduction in the distance between the two ligands.
Fig 7 Backbone superimposition of human (blue) and rat (magenta) AdK The natural ligands (ADA and ATP, blue) and the two AMP molecules (magenta) are shown in bold.
Fig 8 Direct plot of AMP–AMP phosphotransferase activity (as percentage of total activity) in mixtures containing various amounts
of AdK (0.1–4.0 nmol in a final volume of 0.25 mL) and a fixed amount of MK (0.0008 nmol) in the absence of ADA Inset: Scatchard plot.
Trang 8Cooperation between the three enzymes
Our data demonstrate that the AMP–AMP
phospho-transferase reaction occurs by cooperation between
MK and AdK specifically, and is enhanced by ADA;
none of the three enzymes could be substituted by
others
ADA strongly accelerates the AMP–AMP
phospho-transferase reaction, carrying out the coupled reaction
(3) (adenosine fi inosine + NH3) ADA does not
participate directly in ADP synthesis, but its coupled
reaction enables the subtraction of a reaction product,
helping to drive the reaction forwards Moreover, the
exergonic formation of inosine and ammonia from
adenosine fills the gap in the energy balance of
phosphoryl transfer (DG ranges from )4 to )10
-kcalÆmol)1) [14] ADA has a Keq value of
approxi-mately 105 [15], a low Km value and a high efficiency
close to the diffusion-limited rate [16], and is found in
most mammalian tissues [17] It follows that the
AMP–AMP phosphotransferase reaction always
bene-fits from the presence of ADA in vivo
The AMP–AMP phosphotransferase reaction is irre-versible Any attempt at producing AMP by incubat-ing ADP, inosine and NH3 together is ineffective In the absence of ADA, the addition of [14C]adenosine to the mixture does not produce [14C]AMP
Demonstrating the association between AdK and
MK was the key to understanding the mechanism of AMP–AMP phosphotransferase We obtained the following results
(a) No free intermediates were formed during any incubation experiment (that is, no transient dinucleo-tide compound), in contrast with the NADase reaction [18] HPLC, CE and NMR analysis produced no evidence of a reaction intermediate
(b) The existence of an enzyme–phosphate interme-diate, as is formed with 5¢-nucleotidase [19], was ruled out No direct evidence of an enzyme–phosphate com-plex was found Experiments using vanadate or the addition of nucleoside also yielded negative results We found no evidence of AdK–phosphate or MK–phos-phate complex formation in the literature
(c) Microdialysis demonstrated that MK and AdK must be able to associate and work together When they were physically separated, no ADP was formed (d) Electrophoresis and gel filtration experiments excluded covalent or similarly strong interactions (e) In silico simulations suggested that MK contains the active site of the AMP–AMP phosphotransferase reaction, but raised the issue of how the two AMP molecules achieve the correct distance for interaction, and the role of AdK in the reaction
(f) Inhibition experiments confirmed the role of MK
in the reaction Ap5A inhibited MK and AMP–AMP phosphotransferase activities, whereas A134974 only inhibited AdK activity, indicating that the active site
of AdK is not essential for the AMP–AMP phospho-transferase reaction
(g) Kinetic experiments (Fig 8) demonstrated that interactions occurred between the two proteins When
we fixed the amount of MK at a low concentration and increased the concentration of AdK, we obtained
a hyperbolic curve with a saturation trend resembling protein–protein interaction Indeed, isothermal curves, such as the enzyme–substrate curves of Michaelis– Menten, hormone–receptor curves and antigen–anti-body affinity curves, all represent an association between two different molecules and, in the last two cases, between two different proteins [20] In all of these curves, the ordinate values indicate the amount
of dimer formed In the case of Michaelis–Menten plots, the value of V is related to the enzyme–substrate complex; in the case of hormone–receptor curves, the ratio B⁄ Bmax represents the amount of receptor joined
Table 2 Rat liver MK, AdK and AMP–AMP phosphotransferase
specific activities, expressed as lmolÆ(min mg))1 (means ±
stan-dard deviation of eight experiments), were tested in the presence
or absence of 0.25 l M Ap5A (specific inhibitor of MK) or 0.1 n M
A134974 (specific inhibitor of AdK) The mixtures contained 0.2 IU
MK, 0.02 IU AdK and 2 IU adenosine deaminase (the latter only in
the AMP–AMP phosphotransferase assay).
MK activity in the presence of Ap5A 0
MK activity in the presence of A134974 61.42 ± 1.18
AdK activity in the presence of A134974 0
AdK activity in the presence of Ap5A 5.46 ± 0.3
Table 3 MK, AdK, ADA and AMP–AMP phosphotransferase
activi-ties in normal and human colorectal cancer mucosa were assayed.
Partially purified protein preparations (20, 40, 80 and 100 lg,
respectively) were incubated The activities of AdK, ADA and AMP–
AMP phosphotransferase are expressed as lmolÆ(h mg))1, and are
the mean activity ± standard deviation from 10 different patients.
*P < 0.0001.
Trang 9to the hormone with respect to the maximum complex
possible In our case, the ordinate shows the formation
of ADP, which represents the number of dimers
formed between the two proteins In all of these cases,
the association is caused by non-covalent interactions,
such as hydrogen or ionic binding, hydrophobic
inter-actions or van der Waals’ interinter-actions
We conclude that AdK and MK associate through
transient interactions that induce a slight
conforma-tional change in the active site of MK, thereby
bring-ing two already bound molecules of AMP together at
the correct distance for interaction and phosphoryl
transfer from one molecule to the other, ultimately
forming ADP
Physiological role
Our data support the conclusion that the AMP–AMP
phosphotransferase reaction is physiologically
impor-tant The reaction occurs in homogenates and crude
supernatants at pH values close to 7 The apparent Km
value for the substrate is close to its intracellular
con-centration in rat liver [21–23], and is of the same order
of magnitude as that of many enzymes involved in
nucleotide metabolism [24–28] The AdK, MK and
ADA concentrations used in these experiments
coin-cide with the concentrations found in rat liver tissue
[11,12]
Comparing the rates of the AMP–AMP
phospho-transferase, AdK and MK reactions is inappropriate
because the activities, rates and efficiencies of the
three reactions differ in vitro and in vivo according to
the concentrations of their natural substrates (AMP,
ADP, ATP), which vary continuously in different
situations
Under physiological conditions, the AMP–AMP
phosphotransferase reaction may contribute to the fine
regulation of ADP levels Its importance may be
greater under situations associated with ADP and
ATP deficiency, or increased requirements, such as
prolonged physical exertion (when ATP is dramatically
reduced), fructose-induced hyperuricaemia with ATP
depletion [29] and severe nucleotide depletion, as in
rheumatoid arthritis [30] and during cell division The
reaction may play a specific role during transient
ischaemia, anoxia or after reperfusion The behaviours
of AMP, ADP and ATP under such conditions have
been studied extensively and are similar in the liver [1],
heart and brain [10,31] During ischaemia, levels of
ATP and ADP decrease [1,6,10,12,32], whereas those
of AMP increase sharply, reaching up to 2 lmol AMPÆ
g)1 tissue [32] During prolonged ischaemia, ATP
levels decrease to < 10% [1,6,10,32], and ADP levels
decrease to 25–50% of their respective basal concentra-tions [6,32], whereas AMP levels increase by more than
20 times [32] ADP levels are presumably sustained by continuous regeneration, which is unlikely to occur through the classical MK reaction because, under such conditions, the levels of ATP are too low and the levels of AMP are too high to permit classical MK activity Tamura et al [33] reported that liver MK was inhibited by AMP concentrations above 0.5 mm and that the physiological significance of the data were unclear, as this high concentration greatly exceeds the concentration of AMP in rat liver (0.1 mm) In this sit-uation, MK is inhibited and the action of AMP–AMP phosphotransferase prevails, regenerating ADP Gly-colysis and oxidative phosphorylation can regenerate ATP from ADP and Pi; cooperation between these processes and AMP–AMP phosphotransferase will sustain ADP levels and regenerate ATP
The experiments on human cancer mucosa produced interesting results Hypoxia is a well-known feature of locally advanced solid tumours [34], and induces major adaptive responses, such as the production of angio-genic cytokines that promote vascularization and over-expression of the hypoxia-inducible factor-1a gene, which is a classical feature of tumour tissues [35] and was confirmed in our specimens (data not reported) In tumour tissue, AMP levels are more than five times higher than in normal mucosa, whereas the ATP con-centration is more than 10 times lower In contrast, ADP levels remain almost the same (data not reported) Moreover, AMP–AMP phosphotransferase activity was not detectable in normal mucosa, but was substantial in tumour tissue In the same specimens,
MK activity did not vary, whereas ADA and, espe-cially, AdK activities increased Therefore, in hypoxic tissue, the enzyme ratios reach the correct value for the AMP–AMP phosphotransferase reaction The importance of the reaction in tumour tissues will be the subject of future research
We conclude that, under specific conditions, ADP and ATP may be salvaged and restored through the concerted effects of classical ATP formation pathways and the AMP–AMP phosphotransferase network The latter reaction may represent a compensatory mecha-nism for maintaining and increasing ADP to the levels necessary for the restoration of ATP, with the simulta-neous re-utilization of AMP The existence of ATP turnover pathways has been suggested by other authors in studies on MK in creatine kinase-deficient transgenic hearts [36], specifically the existence of alter-native pathways for phospho transfer in the myocar-dium Our experiments indicate that the cooperative action of different proteins may provide fine regulation
Trang 10of metabolic reactions, a poorly understood
phenome-non and an avenue of research that should be explored
further
Experimental procedures
Materials
Male Wistar rats (body weight, 250 g; 9 weeks of age) were
purchased from Harlan Company (S Pietro al Natisone,
Udine, Italy) Nucleosides, nucleotides, bases, enzymes,
analytes and the ATP Bioluminescent Assay Kit
(FLAA-1KT) were obtained from Sigma Life Science (Milan, Italy)
SDS-PAGE reagents and protein assay kits were procured
from Bio-Rad Laboratories s.r.l (Milan, Italy)
Chromato-graphic supports, radioactive compounds and
low-molecu-lar-mass protein molecular mass marker kits were obtained
from GE Healthcare Europe GmbH (Milan, Italy)
N-Hy-droxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl) and
ethanolamine hydrochloride were purchased from Affinity
Sensor (Cambridge, UK) Aquasafe 300 plus was obtained
from Zinsser Analytic (Frankfurt, Germany) HPLC-grade
trifluoroacetic acid was obtained from Carlo Erba Reagenti
SpA (Rodano, Italy) Recombinant enzymes were
pur-chased from ABNOVA Corporation (Taipei, Taiwan) All
other chemicals and HPLC solvents were of analytical
grade and were acquired from Merck KGaA (Darmstadt,
Germany) and J T Baker Italia (Milan, Italy)
Enzyme assays and identification of reaction
products
Enzyme assays
The AMP–AMP phosphotransferase assay mixture
con-tained 4.0 mm cold AMP or 640 kBq [32P]AMP, 50 mm
Bistris (pH 6.5), 1.0 mm MgCl2, up to 0.5 mg of dialysed
crude rat liver supernatant or 1–3 lg of purified fraction
X2 (corresponding to 0.19–0.57 IU of pure MK), fraction
Y2 (corresponding to 0.006–0.018 IU of pure AdK) and
ADA (0.9 IU) in a final volume of 0.25 mL Incubations
were performed at 37C for 15–50 min and stopped with
perchloric acid (neutralized with KOH) or 0.01 mm EDTA;
aliquots were processed by HPLC according to Webster
and Whaun [37] The ammonium content was assayed using
the hypochlorite–phenol Berthelot reaction, according to
Imler et al [38]
One International Unit (IU) of MK, AdK, ADA or
AMP–AMP phosphotransferase was defined as the amount
of enzyme that produced 1 lmolÆmin)1of reaction product
We considered ADP (or ADP + ATP) to be the product
formed by the AMP–AMP phosphotransferase reaction
AMP and all other substrates used in the assay mixture
were purified by HPLC Traces of ATP in the substrates
and enzyme preparations were excluded using the ATP
Bioluminescent Assay Kit CLS II (Roche Diagnostics GmbH, Mannheim, Germany; Sirius Luminometer-Berthold GmbH, Pforzheim, Germany) AdK and ADA were assayed according to Tavernier et al.[39] MK was assayed according to Zhang et al [40]
HPLC analysis
We used a Perkin-Elmer (Monza, Italy) 1020LC Plus system equipped with a ready-to-use prepacked column (Hypersil SAX 5 lm, 150· 4.6 mm; Alltech Italia s.r.l., Segrate, Italy) washed with 5.0 mm ammonium phosphate (pH 2.9) Elution was achieved with 0.5 m ammonium phosphate buffer (pH 4.8) at a flow rate of 1.5 mLÆmin)1, using a linear gradi-ent from 0% to 100% in 10 min The lower limit of detection
of the method was 100 pmol
When [32P]AMP was used, the ADP and ATP peaks were collected and mixed with 10 mL Aquasafe 300 Plus emulsifying scintillator, and the radioactivity was measured using a Packard Model 1500 TriCarb b-counter (Hewlett Packard, Monza, Italy)
Inosine and ADP were identified by multiple means: (a)
by determining the retention times and adding an internal standard; (b) by determining the ultraviolet (UV) spectra in the 210–350 nm range by diode array analysis with a Per-kin-Elmer 235C detector system in line; and (c) by acid hydrolysis of the presumed ADP peak and identification of the products ADP identification was confirmed by testing its ability to act as a substrate for pyruvate kinase (EC 2.7.1.40) [41] After incubation with pyruvate kinase, the HPLC chromatogram of the mixture revealed the disap-pearance of ADP and the formation of an ATP peak, which was identified in the chromatogram using the same criteria as for ADP and inosine
CE analysis All of the above compounds (nucleosides and nucleotides) were also analysed by CE using a Bio-Rad Biofocus 3000 apparatus equipped with a variable wavelength UV detec-tor The assays were performed in an uncoated silica capil-lary (40 cm· 75 lm) with the following operating conditions: 20 mm borate buffer, pH 10, 12 kV and 10 s; hydrostatic load at 25C; 254 nm The compounds were identified by comparing their retention times with those of known internal standards
Enzyme purification and identification by mass spectrometry
The AMP–AMP phosphotransferase reaction occurred only when two or three different enzymes were combined, which were purified according to the following procedures (Table 1)