Evidence of a new phosphoryl transfer system in nucleotide metabolism

Evidence of a new phosphoryl transfer system in nucleotide metabolism Daniela Vannoni1,*, Roberto Leoncini1,*, Stefania Giglioni1, Neri Niccolai2, Ottavia Spiga2, Emilia Aceto1 and 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

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: [email protected] *These authors contributed equally to this work (Received 16 July 2008, revised 24 September 2008, accepted 5 November 2008)

Crude rat liver extract showed AMP–AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adenylate 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 presence 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 interactions 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 phosphotransferase 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.

doi:10.1111/j.1742-4658.2008.06779.x

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 wellknown 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 biological network and cooperate in a reaction not reported previously.

Abbreviations ADA, adenosine deaminase; AdK, adenosine kinase; CE, capillary electrophoresis; MD, molecular dynamics; MK, myokinase.

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These enzymes are well known for their conventional 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: Adenosine þ ATP ! AMP þ ADP (c) Adenosine deaminase (ADA), which catalyses the irreversible reaction:

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.

Adenosine þ H2 O ! inosine þ NH3 The AMP–AMP phosphotransferase reaction is:

AMP þ AMP ! ADP þ inosine þ NH3

AMP þ AMP ! ADP þ inosine þ NH3

resulting from reactions (2) and (3):

ADP and inosine were identified by various methods, including HPLC and diode array analysis; the enzymes responsible for the reaction were identified through a complex purification procedure. Our findings 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 underlying 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 formation 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 [32P]AMP as a substrate, 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 of 32Pi per mole of ADP. These findings indicate that supernatants starting from low-energy precursors catalyse the AMP–AMP phosphotransferase reaction: 272

ð1Þ

AMP þ AMP ! ADP þ adenosine

ð2Þ

Adenosine þ H2 O ! inosine þ NH3 ðADA 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 formation 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 appropriate 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 database using pro found software. The query returned a

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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. norvegicus. 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 recombinant MK and ADA, exhibited AMP–AMP phosphotransferase 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 + 2+ and SO2 and (NHþ 4 ) or had no effect (Ca 4 , Li

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.

Step Common trunk Supernatant P90d DE-52 Not retained (NR) Retained (R) X protein X1 Blue Sepharose X2 Superdex 75 Y protein Y1 AMP Sepharose Y2 Superdex 75

ND ND

ND ND

ND ND

ND ND

ND ND

ND ND

X1 + Y1 Y2 + Y2

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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 component, 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. 2+ PO2 completely eliminated 3 ) on the reaction. Zn 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 conventional 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

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was inefficient because of a lack of substrate. At longer incubation times, when the ADP concentration exceeded the Km value 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 conventional 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: AMP þ AMP

ðAdK;MK;ADAÞ

!

AMP þ AMP

ADP þ inosine þ NH3

ðAdK;MKÞ

!

ADP þ adenosine

ð1Þ 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

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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 associated with the mass spectrometer to a mass accuracy of 0.01%, and can be read directly on the graph.

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 [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 mixture 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 experiments 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

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nmoles

A 9 6

3

0 0 B

10

20

30 40 Minutes

50

60

200

nmoles

150 100 50 0 0

10

20 30 40 50 60 Minutes

Fig. 4. Time course of ADP formation by the AMP–AMP phosphotransferase 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.

phosphoenzyme species. When vanadate was added to the reaction mixture, no inhibition of AMP–AMP phosphotransferase 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 submitted 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 276

Fig. 5. (A) Direct and double reciprocal plot of initial velocities with variable AMP concentration (0.0–6.0 mM) at a constant Mg2+ concentration (1.0 mM). (B) Direct plot of the initial velocities with variable Mg2+ concentration (0.1–1.4 mM) and constant AMP concentration (4.0 mM). 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.

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 natural 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

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A

B

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. 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.

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 Kd value was 1.496 lm, as determined by the Scatchard equation [13].

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.

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 cancerous human colorectal mucosa. The MK activity did not vary, but the AdK and ADA activities were significantly elevated (P < 0.0001) in cancer tissue with respect to the surrounding normal mucosa. AMP– AMP phosphotransferase activity was only detectable in tumour tissue.

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Table 2. Rat liver MK, AdK and AMP–AMP phosphotransferase specific activities, expressed as lmolÆ(min mg))1 (means ± standard deviation of eight experiments), were tested in the presence or absence of 0.25 lM Ap5A (specific inhibitor of MK) or 0.1 nM 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). Assay

Specific activity

MK activity MK activity in the presence of Ap5A MK activity in the presence of A134974 AdK activity AdK activity in the presence of A134974 AdK activity in the presence of Ap5A

62.15 0 61.42 5.42 0 5.46

± 1.59 ± 1.18 ± 0.3 ± 0.3

Table 3. MK, AdK, ADA and AMP–AMP phosphotransferase activities 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 MK ADA

0.010 ± 0.004 25.89 ± 9.72 0.441 ± 0.156

0.021 ± 0.009* 22.41 ± 7.81 0.657 ± 0.253*

Discussion Cooperation between the three enzymes Our data demonstrate that the AMP–AMP phosphotransferase 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 phosphotransferase 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 approximately 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 benefits from the presence of ADA in vivo. 278

The AMP–AMP phosphotransferase reaction is irreversible. Any attempt at producing AMP by incubating 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 dinucleotide 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 intermediate, as is formed with 5¢-nucleotidase [19], was ruled out. No direct evidence of an enzyme–phosphate complex was found. Experiments using vanadate or the addition of nucleoside also yielded negative results. We found no evidence of AdK–phosphate or MK–phosphate 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 phosphotransferase 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–antibody 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

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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 interactions or van der Waals’ interactions. We conclude that AdK and MK associate through transient interactions that induce a slight conformational change in the active site of MK, thereby bringing 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 important. 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 concentration 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 coincide with the concentrations found in rat liver tissue [11,12]. Comparing the rates of the AMP–AMP phosphotransferase, 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

Formation of ADP from AMP

decrease to 25–50% of their respective basal concentrations [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 situation, MK is inhibited and the action of AMP–AMP phosphotransferase prevails, regenerating ADP. Glycolysis 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 angiogenic cytokines that promote vascularization and overexpression 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 concentration 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, especially, 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 mechanism for maintaining and increasing ADP to the levels necessary for the restoration of ATP, with the simultaneous 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 alternative pathways for phospho transfer in the myocardium. Our experiments indicate that the cooperative action of different proteins may provide fine regulation

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of metabolic reactions, a poorly understood phenomenon and an avenue of research that should be explored further.

Experimental procedures

Bioluminescent Assay Kit CLS II (Roche Diagnostics GmbH, Mannheim, Germany; Sirius LuminometerBerthold GmbH, Pforzheim, Germany). AdK and ADA were assayed according to Tavernier et al.[39]. MK was assayed according to Zhang et al. [40].

Materials

HPLC analysis

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 (FLAA1KT) 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). Chromatographic supports, radioactive compounds and low-molecular-mass protein molecular mass marker kits were obtained from GE Healthcare Europe GmbH (Milan, Italy). N-Hydroxysuccinimide, 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 purchased 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).

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 gradient 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 Perkin-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 disappearance of ADP and the formation of an ATP peak, which was identified in the chromatogram using the same criteria as for ADP and inosine.

Enzyme assays and identification of reaction products Enzyme assays The AMP–AMP phosphotransferase assay mixture contained 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 37 C 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)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

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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 detector. The assays were performed in an uncoated silica capillary (40 cm · 75 lm) with the following operating conditions: 20 mm borate buffer, pH 10, 12 kV and 10 s; hydrostatic load at 25 C; 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).

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(a) Common trunk. Rat livers were obtained from animals 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. The pure final protein was named X2. (c) Y2 fraction purification. A DE-52 column was developed with 10 column volumes of a linear gradient (0–100%) of buffer A spiked with 0.1 m KCl. The eluted and concentrated fractions were named fraction Y and applied to an AMP-Sepharose column (1.6 · 2.5 cm), equilibrated with buffer B at a flow rate of 3 mLÆh)1 and washed with buffer B containing 5 mm adenosine. The eluted proteins were washed several times with buffer C, concentrated (fraction Y1) and submitted to gel filtration chromatography as described for X2. The pure final protein was named Y2. (d) ADA purification. ADA was purified from P90d according to Schrader et al. [42], with slight modifications (data not shown). All of the chromatographic procedures were performed using a high-performance AKTA Purifier System (GE Healthcare) and a unicorn Control System (Version 5.10) as the control platform. Total proteins were assayed according to Bradford [43] using BSA as a standard. The purity of the fractions was monitored by 12% SDS-PAGE under non-reducing conditions [44], and the gels were developed with silver nitrate stain. X2 and Y2 proteins were identified by mass spectrometry, performed at the International Centre for Mass Spectrometry Services, University of Naples, or at the Centre for Structural Determinations, University of Siena. A 200 lg portion of X2 or Y2 fractions was desalted by RP-HPLC on a Phenomenex C4 column (25 · 0.46 cm, 5 lm), and eluted with 0.1% trifluoroacetic acid (solvent A) and 0.07% trifluoroacetic 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

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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 MALDITOF 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 lL of a 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 MALDI spectra were used for a database search employing profound 4.10.5 software downloaded from the web (http:// prowl.rockefeller.edu/). Unknown proteins were identified by determining the probability score associated with each putative candidate.

Expression of AdK in recombinant E. coli An aliquot of human AdK clone 911, obtained from the human liver cDNA kZAP library in a pET-24b vector (kindly donated by J. Spychala, University of North Carolina), was used to express AdK in E. coli BL 21[DE3] cells Induction was performed for 4–6 h in the presence of 1 mm isopropyl thio-b-d-galactoside. Recombinant AdK protein was obtained from the cells by lysing the bacterial pellet in 10 volumes of 50 mm Tris ⁄ HCl (pH 7.5) containing 1 mm EDTA, 1 mm dithiothreitol and 1 mgÆmL)1lysozyme, using three freeze–thaw cycles in liquid nitrogen. The lysate was ultracentrifuged and the supernatant was purified as described previously [45].

Reaction mechanism studies – cooperation between AdK and MK Micro-equilibrium dialysis experiments The micro-equilibrium dialysis apparatus (Micro-Equilibrium Dialyser, Harvard Apparatus Ltd., Edenbridge, UK) consisted of two chambers for small samples of equivalent volume (0.1 mL), separated by a cellulose acetate membrane (5 kDa cut-off) impermeable to proteins. Each chamber contained AMP, Bistris and MgCl2, as reported for the standard assay mixture, in a final volume of 0.1 mL. Suitable

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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 chambers 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 determined 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 phosphotransferase assay mixtures (2 mL) spiked with 2H2O were placed in 10 mm diameter tubes and incubated overnight 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 broadening to improve the signal-to-noise ratio.

Enzyme–phosphate intermediate trapping experiments We used the procedure described by Baiocchi et al. [19] to study cytosolic 5¢-nucleotidase phosphotransferase. The reaction mixture contained 0.12 nmol pure MK, 0.14 nmol pure AdK, 0.11 nmol pure ADA, 1.5 mm [32P]AMP (3 CiÆmmol)1), 2.5 mm AMP, 50 mm Bistris ⁄ HCl buffer (pH 6.5) and 1 mm MgCl2 in a final volume of 50 lL. After 15 and 30 min at 37 C, the reaction was stopped by the addition of 12 lL 20% glycerol ⁄ 4% SDS in 125 mm Tris ⁄ HCl (pH 7.4). The samples were loaded onto a 12% SDS-PAGE gel, as described previously. The proteins were electroblotted onto a nitrocellulose membrane (0.45 mm) for 1 h at 100 V. After 4 days at )80 C, 32P-labelled proteins were identified by autoradiography. Another approach used to ascertain the formation of phosphoryl enzyme intermediates consisted of spiking the reaction with vanadate, which inhibits the enzymes that catalyse phosphoryl transfer through the formation of phosphoryl enzyme intermediates [46], or with nucleosides, such as [14C]adenosine, [14C]guanosine or [14C]inosine. If covalent phosphoenzyme 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 prepacked Superdex 75 HR 10 ⁄ 30 column equilibrated and

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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 (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) was used to select suitable templates for homology modelling of AdK and MK structures. The modelling procedure was performed using deepview v.3.5 [48] and structure validation was obtained with procheck v.3.5.4 [49]. To find the best templates, we submitted the sequence of two proteins (SwissProt ID code Q5EBC5 for AdK and Q642G1 for MK) (http://www.expasy.ch/sprot/) to blast. The corresponding human proteins, exhibiting 92% and 90% identity, respectively, were used as templates by retrieving their structures from the Protein Data Bank (http:// www.rcsb.org/pdb/): ID code 3AdK for AdK [50] and PDB ID code 1BX4 for MK [51]. The initial docking simulation of AMP into the protein active sites was performed by manually placing the molecule at different locations. autodock software v.2.4 [52] was then used to obtain the conformation with the lowest potential energy. The protein complexes of rat AdK and MK were placed at the centre of a rectangular box that was filled with 8450 and 14 203 simple point charge water molecules, respectively, and one Cl) and four Na+ ions, to obtain a net charge of zero. To relax the configuration of the solvent, a steep descent minimization was performed. All simulations were run using the gromacs v.3.2 bimolecular package [53]. The following protocol was used for the MD simulation. To equilibrate the system, the temperature was increased stepwise from 50 K to the target temperature of 300 K in a 40 ps run. This was followed by a 1 ns production run at constant temperature. A trajectory frame dumped every picosecond. The protein and water were separately coupled to a temperature bath with a relaxation constant of 0.5 ps. The pressure was maintained at 101.325 kPa by coupling to a pressure bath with a relaxation constant of 0.5 ps.

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 Kd value was determined according to the Scatchard equation.

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Inhibition experiments Three series of experiments were performed in which we tested MK, AdK or AMP–AMP phosphotransferase activity with or without the addition of 0.25 mm Ap5A (P1,P2-diadenosine-5¢-pentaphosphate), a specific competitive 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 specimens from 10 patients with cancer (eight men, two women; age, 46–74 years) hospitalized at the Department of General Surgery, University of Siena. The clinical histories of the patients were known; no patient had received chemotherapy or radiation therapy prior to surgery. Samples of tumour tissue and normal mucosa located more than 10 cm from the tumour were obtained from all patients. The fresh surgical biopsies (70 mg) were immediately frozen in liquid nitrogen and stored at )80 C until assay. After thawing, the samples were rinsed of residual blood with cold 10 mm Hepes (pH 7) spiked with 0.1 mm MgCl2, homogenized in the same buffer using an Ultra-Turrax blender (Ika Werke GrbH, Staufen, Germany) and centrifuged at 65 000 g for 30 min in a Beckman L7-65 ultracentrifuge (Beckman Coulter SpA, Cassina de Pecchi, Italy) at 4 C. The clear supernatant was partially purified on a CM Sepharose column (1.6 cm · 1 cm), equilibrated with the same buffer and eluted at a flow rate of 0.1 mLÆmin)1 to remove any interfering contaminants. The non-retained fraction contained MK and AdK activities. ADA was eluted using a linear gradient with the same buffer spiked with 2 m NaCl in five column volumes. MK, AdK, ADA and AMP–AMP phosphotransferase activities were assayed using the mixtures reported previously for rat liver, incubating 20, 40, 80 and 100 lg, respectively, of partially purified protein preparations.

Statistical analysis Data were analysed by the Mann–Whitney U-test using prism 4.0 by GraphPad Software Inc. (San Diego, CA, USA). Differences yielding a P value of < 0.05 were considered to be significant. Kinetic data (Km and Kd) 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 ([email protected]), for assistance in editing

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the manuscript. This work was supported by grants from the University of Siena and Italian Ministry of Education.

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