Evidence of a new phosphoryl transfer system in
nucleotide metabolism
Daniela Vannoni
1,
*, Roberto Leoncini
1,
*, Stefania Giglioni
1
, Neri Niccolai
2
, Ottavia Spiga
2
, Emilia
Aceto
1
and Enrico Marinello
1
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

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.
FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS 271
These enzymes are well known for their conven-
tional reactions:
(a) Adenylate kinase, also called myokinase (MK),
which catalyses the reversible reaction:
AMP þ ATP $ ADP
(b) Adenosine kinase (AdK), which catalyses the
irreversible reaction:

tion of two products with retention times of 1.3 and
7.1 min, which were identified as inosine and ADP,
respectively (Fig. 1). Using purified [
32
P]AMP as a sub-
strate, ADP formation was consistent with the transfer
of
32
P between two molecules of [
32
P]AMP to form
[
32
P]ADP[a,bP]. Thus, starting from [
32
P]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 of
32
P
i
per mole of ADP.
These findings indicate that supernatants starting
from low-energy precursors catalyse the AMP–AMP

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.
Formation of ADP from AMP D. Vannoni et al.
272 FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS
significant 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).

value for
AMP was 0.8 mm (Fig. 5A). Mg
2+
was essential for
the AMP–AMP phosphotransferase reaction, with the
optimal concentration in the range 0.8–1.5 mm. The
apparent K
m
value of Mg
2+
was 0.35 mm (Fig. 5B).
Certain ions added to the incubation mixture inhibited
(NH
þ
4
,Li
+
and SO
2À
4
) or had no effect (Ca
2+
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

PO
2À
3
) on the reaction. Zn
2+
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 [
32
P]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

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.
Formation of ADP from AMP D. Vannoni et al.
274 FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS
predominant, 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 [
14
C]adenosine to
the incubation mixture, no [
14
C]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

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

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 mM). (B) Direct plot of the initial velocities with
variable Mg
2+
concentration (0.1–1.4 mM) 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.
Formation of ADP from AMP D. Vannoni et al.
276 FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS
gradually 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 K
d
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. Ap

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.
D. Vannoni et al. Formation of ADP from AMP
FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS 277
Discussion
Cooperation 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 + NH
3
). 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 )4to)10 -
kcalÆmol
)1
) [14]. ADA has a K
eq

(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. Ap
5
A 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

MK activity in the presence of A134974 61.42 ± 1.18
AdK activity 5.42 ± 0.3
AdK activity in the presence of A134974 0
AdK activity in the presence of Ap
5
A 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.
Activity Normal mucosa Tumour mucosa
AdK 0.010 ± 0.004 0.021 ± 0.009*
MK 25.89 ± 9.72 22.41 ± 7.81
ADA 0.441 ± 0.156 0.657 ± 0.253*
Formation of ADP from AMP D. Vannoni et al.
278 FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS
to 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’ interactions.
We conclude that AdK and MK associate through
transient interactions that induce a slight conforma-

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

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
D. Vannoni et al. Formation of ADP from AMP
FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS 279
of 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

)1
of 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 [
32
P]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).

280 FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS
(a) Common trunk. Rat livers were obtained from ani-
mals housed at 20 °C, fed a standard laboratory diet and
killed by decapitation after a 12 h fast. The livers were
homogenized (15%) in buffer A (50 mm Tris ⁄ HCl, pH 7.5,
containing 0.1 mm dithiothreitol) and 0.25 m sucrose, and
centrifuged at 240 000 g for 60 min. The supernatant was
precipitated with ammonium sulfate (65–90% saturation)
and dialysed overnight (P90d).
(b) X2 fraction purification. The P90d fraction was applied
to a DE-52 cellulose column (5 · 6.5 cm; flow rate, 60 mLÆh
)1
)
and washed with three column volumes of buffer A.
Non-retained proteins were concentrated and washed with
buffer B (10 mm potassium phosphate, pH 7.0, with 0.1 mm
dithiothreitol) using centrifugal filters (CentriconPlus 20:
Fraction X; Millipore SpA, Vimodrone, Italy), and applied
to a Blue-Sepharose HiTrap column (5 mL) equilibrated with
buffer B and eluted using a step gradient with buffer B plus
KCl at a flow rate of 3 mLÆh
)1
. Fractions eluted with 0.5 m
KCl were pooled, washed several times with buffer C (10 mm
Tris ⁄ HCl, pH 7.5, with 0.1 mm dithiothreitol and 0.15 m
KCl), concentrated (fraction X1), submitted to gel filtration
chromatography using a prepacked Superdex 75 HR 10 ⁄ 30
column, and eluted with buffer C at a flow rate of 30 mLÆh
)1
.

with 0.1% trifluoroacetic acid (solvent A) and 0.07% trifluo-
roacetic acid in 95% acetonitrile (solvent B) using a linear
gradient of 20–95% solvent B over 15 min. The elution
was monitored at 220 nm, and the fraction was collected
manually and injected directly into the electrospray mass
spectrometry apparatus. Analysis was performed using a ZQ
single quadrupole mass spectrometer equipped with an
electrospray ion source (Micromass, Manchester, UK) and a
Harvard syringe pump, with a flow rate of 5 lLÆmin
)1
.
Spectra were recorded by scanning the quadrupole (10 sÆper
scan). Data were acquired and processed using masslynx
software (Micromass). Mass scale calibration was performed
with multiply charged ions from a separate injection of horse
heart myoglobin (average molecular mass, 16 951.5 Da).
Trypsin digestion of the proteins was performed overnight in
0.4% ammonium bicarbonate (pH 8.5) at 37 °C using an
enzyme to substrate ratio of 1 : 50 (w ⁄ w). Trypsin–peptide
mixtures were analysed with a Voyager DE-PRO MALDI-
TOF mass spectrometer (Applied Biosystems, Monza, Italy)
equipped with a Reflectron analyser. The mass range was
calibrated using a mixture of five standard peptides provided
by the manufacturer. A 1.0 lL sample (approximately) was
applied to a sample slide and mixed with 1.0 lLofa
10 mgÆmL
)1
la-cyano-4-hydroxycinnamic acid solution in
acetonitrile with 0.2% trifluoroacetic acid (70 : 30, v ⁄ v)
before air drying. Peptide mass values recorded in the

, as reported for the stan-
dard assay mixture, in a final volume of 0.1 mL. Suitable
D. Vannoni et al. Formation of ADP from AMP
FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS 281
amounts of AdK and MK were placed in the same chamber,
or one in either chamber separated by the membrane, in the
presence or absence of ADA. In all cases, the apparatus was
incubated at 37 °C for 1, 24 or 36 h. Reactions in both cham-
bers were stopped with 0.01 mm EDTA and analysed by
HPLC.
Detection of intermediates by HPLC, CE and NMR
analysis
The presence of intermediate reaction products was deter-
mined by HPLC and CE, as described previously, and by
31
P NMR spectra, obtained using an FT spectrometer
(model AMX-400, Bruker BioSpin, Milan, Italy) operating
at 161.923 MHz. In these experiments, AMP–AMP phos-
photransferase assay mixtures (2 mL) spiked with
2
H
2
O
were placed in 10 mm diameter tubes and incubated over-
night inside the magnet at 37 °C. The spectra were collected
as an accumulation of 128 scans with a 0.5 s repetition
time. Analysis was performed on an SGI data station, and
acquisition and processing parameters were identical in all
experiments. Spectra were obtained with 5 Hz line broaden-
ing to improve the signal-to-noise ratio.

14
C]guanosine or [
14
C]inosine. If covalent phospho-
enzyme intermediates (enzyme–phosphate–nucleoside) are
formed in the principal reaction, an exchange reaction of
nucleotide fi nucleoside is possible; then, a new nucleotide
is released and the principal reaction is inhibited [47].
Gel filtration and SDS-PAGE
Gel filtration experiments were performed using a pre-
packed Superdex 75 HR 10 ⁄ 30 column equilibrated and
eluted with 10 mm potassium phosphate buffer (pH 7.0)
and 0.15 m NaCl at a flow rate of 0.5 mLÆmin
)1
. In some
experiments, 0.1 mm AMP was added to the eluent
buffer. Twenty micrograms of pure AdK, MK and ADA
were incubated for 0 and 60 min at 37 °C and loaded on
to the column. Under these conditions, proteins and
reaction products were separated and detected in the
same run. SDS-PAGE was performed as described
previously.
Docking simulation studies
blast software ( />was used to select suitable templates for homology model-
ling of AdK and MK structures. The modelling procedure
was performed using deepview v.3.5 [48] and structure vali-
dation was obtained with procheck v.3.5.4 [49]. To find
the best templates, we submitted the sequence of two pro-
teins (SwissProt ID code Q5EBC5 for AdK and Q642G1
for MK) ( to blast. The corre-

Kinetic experiments
In the kinetic experiments, various amounts of pure AdK
(0.4–1.6 lm) were incubated with a fixed amount of MK
(0.0032 lm); ADA was not added to the assay mixture.
The K
d
value was determined according to the Scatchard
equation.
Formation of ADP from AMP D. Vannoni et al.
282 FEBS Journal 276 (2009) 271–285 ª 2008 The Authors Journal compilation ª 2008 FEBS
Inhibition experiments
Three series of experiments were performed in which we
tested MK, AdK or AMP–AMP phosphotransferase activ-
ity with or without the addition of 0.25 mm Ap
5
A
(P
1
,P
2
-diadenosine-5¢-pentaphosphate), a specific competi-
tive inhibitor of MK [54], or 0.1 nm A134974, a specific
competitive inhibitor of AdK [55], or both.
AMP–AMP phosphotransferase reaction in human
colorectal mucosa from cancer patients
Tissue was obtained from surgically resected colon speci-
mens from 10 patients with cancer (eight men, two women;
age, 46–74 years) hospitalized at the Department of Gen-
eral Surgery, University of Siena. The clinical histories of
the patients were known; no patient had received chemo-

sidered to be significant. Kinetic data (K
m
and K
d
) were
evaluated using the same software.
Acknowledgements
We thank Professor J. Spychala, University of North
Carolina, for the human AK clone 911, and Paul
Kretchmer PhD, Managing Director San Francisco
Edit (), for assistance in editing
the manuscript. This work was supported by grants
from the University of Siena and Italian Ministry of
Education.
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