31P Saturation Transfer Spectroscopy Predicts DifferentialIntracellular Macromolecular Association of ATP and ADP inSkeletal Muscle*□S

Received for publication, July 15, 2010, and in revised form, September 21, 2010 Published, JBC Papers in Press, September 29, 2010, DOI 10.1074/jbc.M110.164665

Christine Nabuurs‡, Bertolt Huijbregts‡, Be Wieringa§, Cees W. Hilbers¶, and Arend Heerschap‡1

From the Departments of ‡Radiology and §Cell Biology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen and the¶Laboratory of Physical Chemistry, Faculty of Science, Radboud University Nijmegen, 6525 GA Nijmegen, The Netherlands

The kinetics of phosphoryl exchange involving ATP andADPhave been investigated successfully by in vivo 31P magnetic res-onance spectroscopy using magnetization transfer. However,magnetization transfer effects seen on the signals of ATP alsocould arise from intramolecular cross-relaxation. This relax-ation process carries information on the association state ofATP in the cell. To disentangle contributions of chemicalexchange and cross-relaxation tomagnetization transfer effectsseen in 31Pmagnetic resonance spectroscopy of skeletal muscle,we performed saturation transfer experiments on wild type anddouble-mutant mice lacking the cytosolic muscle creatinekinase and adenylate kinase isoforms. We find that cross-relax-ation, observed as nuclearOverhauser effects (NOEs), is respon-sible for magnetization transfer between ATP phosphates bothinwild type and inmutantmice. Analysis of 31P relaxation prop-erties identifies these effects as transferred NOEs, i.e. underly-ing this process is an exchange between free cellular ATP andATP bound to slowly rotating macromolecules. This explainsthe �-ATP signal decrease upon saturation of the �-ATP reso-nance. Although this usually is attributed to �-ADP7 �-ATPphosphoryl exchange, we did not detect an effect of thisexchange on the �-ATP signal as expected for free [ADP],derived from the creatine kinase equilibrium reaction. Thisindicates that in resting muscle, conditions prevail that preventsaturation of�-ADP spins and puts into question the derivationof free [ADP] from the creatine kinase equilibrium.We presenta model, matching the experimental result, for ADP 7 ATPexchange, in which ADP is only transiently present in thecytosol.

Phosphoryl exchange reactions are the backbone of energytransduction in living systems (1). The possibility to assess therates of some key phosphoryl exchange reactions in vivo is aunique property of magnetization transfer (MT)2 methods in

31P MR spectroscopy (2, 3). These methods involve specificmagnetic labeling of a phosphate spin system and subsequentobservation of exchange of its members with other phosphatespin systems. In MT studies performed on muscles and brain,�-ATP phosphate has played a central role. This phosphateparticipates in multiple exchange reactions, most prominentlythose catalyzed by creatine kinases (CK) in these tissues, butalso by adenylate kinases (AK), nucleotide diphosphate kinases,and ATPases. In skeletal muscle, magnetic labeling by selectivesaturation of the �-ATP phosphorus signal is known to reducethe intensity of the signals of phosphocreatine (PCr) and inor-ganic phosphate (Pi). The decreases result from ATP-produc-ing chemical exchange reactions

PCr -|0kCK,for

kCK,rev

�ATP -|0kPi,for

kPi,rev

Pi

REACTION 1

Upon saturation of the �-ATP signal, CK activity reduces thePCr signal intensity, whereas the Pi signal intensity decays pro-portional to the activity of mitochondrial F1F0-ATPase and thecombination of glycolytic enzymes glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and phosphoglycerate kinase (4).Apart from the reduction of the PCr and Pi signal intensity,

the �-ATP signal intensity also decreases upon irradiation ofthe �-ATP spins (3, 5–14). This effect, which has received rel-atively little attention, is usually attributed to contributionsfrom several phosphoryl exchange pathways. As �-ATP and�-ADP spins resonate at nearly the same frequency, the �-ADPspins are easily co-saturated with the �-ATP spins. This mayaffect the �-ATP signal according to the reverse CK reaction(ATP � Cr 3 ADP � PCr � H�) and even more so if AK,F1F0-ATPase and/or glycolytic enzymes also contribute to theATP to ADP conversions involving the chemical exchange

� � ATP 9|=kADP,for

kADP,rev

� � ADP

REACTION 2

in which kADP,for and kADP,rev represent pseudo first order rateconstants, meaning that they not only represent the intrinsicrate constant but are also determined by enzyme and substrateconcentrations. Unless stated otherwise, this holds for all equa-tions used in this study.

* This work was supported by Netherlands Organization for ScientificResearch Grants 834.04.007 and 40-00506-98-06021) and the Prinses Beat-rixfonds (Grant WAR06-0217).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental text and Fig. S1.

1 To whom correspondence should be addressed: Prof. Geert Grooteplein 10,6500HB Nijmegen, The Netherlands. E-mail: A.Heerschap@rad.umcn.nl.

2 The abbreviations used are: MT, magnetization transfer; CK, creatinekinase(s); AK, adenylate kinase(s); M-CK, muscular cytosolic CK; MAK�/�,double knockout for M-CK and AK1; Cr, creatine; PCr, phosphocreatine;CSA, chemical shift anisotropy; trNOE, transferred NOE.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 51, pp. 39588 –39596, December 17, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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A detailed investigation of the reduction of the �-ATP signalhas been carried out by Le Rumeur et al. (5). They studied thekinetics of ATP to ADP �-phosphoryl exchange in resting andcontracting rat skeletal muscle. An interesting conclusion fol-lowing from their work was that deriving the �-phosphorylconversion from the reduction of the �-ATP signal in restingmuscle highly underestimates the expected value that isobtained from the �-ATP-PCr exchange in the CK reaction. Infact, a more complex picture applies as in addition to chemicalexchange, the effect on the �-ATP signal could be caused bycross-relaxation leading to nuclear Overhauser enhancement(NOE).In this study, we aimed to assess contributions from cross-

relaxation and from chemical exchange to the reduction of the�-ATP signal. This is of importance first for a proper under-standing of this effect but also because the extent of these con-tributions critically depends on the physical state of ATP andADP in vivo. Finally, the results also constitute a test for thevalidity of the common practice to calculate free ADP concen-trations in skeletal muscle from the CK reaction, whichassumes that this reaction is at (near-)equilibrium (15–17). Toassist in the distinction between cross-relaxation and chemicalexchange, we compared MT effects in 31P MR spectroscopy ofskeletal muscle of wild type mice and mutant mice, which lackcytosolic CK and AK activities (only mitochondrial CK and AKremain) and therefore have strongly reduced phosphoryl trans-fer capacity (18).By carefully analyzing the 31P relaxation properties of all

three phosphates in ATP, we show that the reduction of the�-ATP signal is not caused by phosphoryl exchange reactionsbut by so-called transferred NOEs brought about by the inter-action of ATP with slowly tumbling cellular components. Ourexperiments also indicate that in resting skeletal muscle, con-ditions prevail in which the �-phosphate resonance of ADPparticipating in the �-phosphoryl exchange cannot be satu-rated. To account for these experimental results, we proposethat ADP in muscle cells at rest is associated with immobile(slowly tumbling) structures with a solid state-like characterand participates in the ADP 7 ATP exchange reaction,wherein it is only transiently freely present in the cytosol.

EXPERIMENTAL PROCEDURES

Animal Subjects and in Vitro Samples—In vivo MR experi-ments were performed on compound mutant mice lacking thegenes for both the cytosolic isoforms of muscular CK (M-CK)and AK (AK1) (MAK�/�: n � 13, 8.5 � 5.0 months, 29.5 �4.0 g) and the wild type littermates (WT: n � 10, 10.1 � 3.0months, 27.7 � 3.5 g). Residual phosphoryl transfer capacitystill exists, resulting from the presence of mitochondrial iso-forms ScCKmit and AK3 in muscles of these animals (account-ing for �8% and �1–2% of wild type activities, respectively(18)). The generation ofMAK�/�mice has been described else-where (18). All experimental procedures were approved by thelocal animal ethics committee. In vitromeasurementswere per-formed on a solution containing: 7 mM ATP, 10 mM MgCl, 35mMPCr, and 1mMEDTA in 100mMTris-HCl buffer, pH� 7.5.NMR Experiments—31PMR experiments on murine skeletal

muscle were carried out at 7.0 tesla, in a 120-mm horizontal

bore, magnet (Magnex Scientific, Abingdon, UK) interfaced toan MR spectrometer (MR Solutions, Surrey, UK) operating at121.53 MHz for 31P. MR spectra were acquired from the hindleg of the mice with a 9-mm diameter solenoid coil (19). A lowpower continuous wave pulse (�B2 � 320 radians/s) wasapplied prior to the acquisition pulse to saturate signals selec-tively for variable durations (t): 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, and5.0 s. The spectra were acquired using 64 averages at a repeti-tion time of 7 s. Signal decreases in PCr, Pi, �-ATP, and �-ATPwere determined upon saturation of the �-ATP/�-ADP signals.Similarly, signal decreases in �-ATP, �-ATP, and �-ATP wereobserved when positioning the irradiation pulse at the PCr,�-ATP, and �-ATP resonances, respectively. For each experi-ment, control spectra with the irradiation pulse at mirror fre-quencies were acquired to correct for radio frequency bleeding.DataAnalysisMRSpectra—Signal integralswere fitted in the

time domain with the program MRUI (AMARES fitting algo-rithm) using Gaussian line shapes for Pi, PCr, �-ATP, �-ATP,NADPH, and �-ATP at 4.8, 0, �2.5, �7.5, �8.2, and �16.2ppm, respectively. The line widths of Pi and NADPH signalswere constrained to 1.5 and 1.0 times that of PCr. The PCr andPi signal intensities were scaled to the �-ATP signal and cor-rected for T1 saturation. Tissue concentrations were calculatedassuming thatATP levels of bothWTandMAK�/�mice in thisstudy were 7.8 � 0.08 mM (19). Tissue pH was obtained fromthe chemical shift difference between PCr and Pi (20). Mono-exponential functions were fitted to the time dependent decaysobtained in the relaxation measurements using a non-linearleast square method: the Levenberg-Marquardt algorithm(GraphPad Prism, San Diego CA).

RESULTS

High Energy Phosphate Levels and CK Activity—In vivo 31PMR spectra ofWTandMAK�/� skeletalmuscle acquiredwith-out the application of saturation pulses look very similar (Fig.1). Analysis of the resonance positions and intensities showsthat the pH and the levels of phosphate-containingmetabolitesinMAK�/� mice are virtually equal to those inWT littermatesexcept for slightly decreased PCr levels (Table 1). Thus, station-ary conditions with near normal levels of phosphorylation of Crare achieved in MAK�/� mice despite the lack of cytosolicM-CK.However, the rate at which this steady state is reached isprofoundly different. This point is best illustrated by thesmaller reduction of the PCr signal upon selective saturation ofthe �-ATP spins in the MAK�/� mice. The PCr signal declinesas a function of the length of the saturation pulse (Fig. 2)according to

MzA�t� � MzA0

MzA0 �

kfor

�A � kfor�e���A � kfor�t � 1� (Eq. 1)

in whichMzA0 is the PCr equilibrium magnetization,MzA(t) is

the PCr magnetization after the saturation pulse of duration t,�A is the is the relaxation constant of the PCr spins, A, and kforis the pseudo first order rate constant in forward direction,PCr3 ATP, see reviews (2, 3).It is clear that the absence of cytosolic M-CK activity in the

mutant muscle results in strongly reduced MT effects on the

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PCr signal intensity when compared with MT effects in WT.The time dependence of the WT PCr signal could be fittedwith a monoexponential function, indicating that the effect ofthe possible cross-relaxation between the phosphorous and

proton spins in PCr is negligible(21), as is tacitly assumed in the der-ivation of Equation 1. The pseudofirst order rate constants (kCK,for) ofthe CK-mediated exchange in for-ward direction (PCr 3 ATP)obtained in this way reflect thestrongly decreased exchange in themutant mice when compared withthat of theWT littermates (Table 2).MT Effects in ATP in the Presence

and Absence of M-CK and AKActivity—To determine the flux viathe CK-mediated reaction fromdecreases in PCr signal intensity,the �-ATP resonance is selectivelysaturated in in vivo 31P saturationtransfer applications. When weapplied this procedure, we observedthat the PCr signal also decreasedupon saturation of the �-ATP sig-

nal. This unanticipated behavior was only found for the WTmice; in the mutants, the PCr remained unaltered (Fig. 3). Thissuggests participation of the CK-mediated reaction in this MTeffect in the WT mice. However, between �-ATP and PCr, nodirect phosphate exchange does occur. Because the absence ofthis effect in MAK�/� mice eliminates off-resonance satura-tion as a cause of this effect, the reducedPCr signals inWTmicecan only be ascribed to a chemically relayed NOE, i.e. cross-relaxation between �- and �-ATP spins followed by CK-medi-ated exchange.Indeed, steady state saturation of the �-ATP signal leads to a

signal reduction of 22 � 5% not only at the �-ATP resonancebut also at the �-ATP signal intensity (25% � 8% for WT). Tocomplete this picture, we also irradiated the �-ATP and �-ATPresonances and observed similarMT effects on the �-ATP spinsystem. Within the experimental accuracy of our approach, allthese effects were of similarmagnitude, ranging from 21 to 26%(Fig. 4). This symmetry is expected if theMT effects are causedby dipolar cross-relaxation between the phosphorous spins,leading to NOE effects. NOE involvement is corroborated bythe MT to the �-ATP signal upon irradiation of the �-ATPsignal, which rules out the contribution of chemical exchangereactions to these magnetic transfer effects. In this respect, it isworth mentioning that the decrease in signal intensity uponsaturation of directly neighboring phosphates was equal forWT and MAK�/� skeletal muscle. Hence M-CK and AK1activity cannot be held responsible for the MT effects betweenthe phosphates of ATP. In contrast to the NOE effects thatoccur between directly neighboring phosphorous spins, themagnetization transfer between �- and �-ATP spins (onlyinvestigated for WT mice) turned out to be very small (�-ATPdecreased with 5� 4% upon �-ATP saturation). This can easilybe explained by the larger distance (4.5 Å) between the twoouter phosphates.Absence of NOE in ATP in Vitro—To further investigate the

results, we set out to measure NOE effects between the phos-phates ofATP for a 7mMATP solution, butwithout success.No

FIGURE 1. 31P MR spectra of skeletal muscle of WT and MAK�/� mice recorded in the presence andabsence of saturation of the �-ATP(/�-ADP) signals. For both groups, the spectra on the left are unper-turbed; in the spectra on the right-hand side, the �-ATP resonance at �2.5 ppm is saturated by a pulse of 5-sduration (gray arrow). The second spectrum from the right (WT mice, saturated �-ATP/(�-ADP)) shows a largedecrease of the PCr signal intensity as a result of the saturation of the �-ATP resonance, whereas the secondspectrum on the far right (MAK�/� mice, saturated �-ATP/(�-ADP)) shows only a very small perturbation as aresult of the saturation of the �-ATP signal. Both groups show decreases in �-ATP signal and a small decreasein Pi.

FIGURE 2. Reduction of the PCr signal intensity upon selective saturationof the �-ATP resonance in WT (f) and MAK�/� (F) mice as a function ofthe duration of the saturation pulse. MAK�/� mice show a clearly reducedMT effect on the PCr resonance when compared with WT littermates, whichimplies a lower forward rate through the CK reaction. Control spectra with theirradiation pulse at mirror frequency (at 2.5 ppm) were acquired to correct forradio frequency bleeding; i.e. the magnetization of PCr upon saturation of the�-ATP was corrected for off-resonance effects by subtraction of the magneti-zation of PCr (M0). Error bars indicate mean � S.E.

TABLE 1Tissue concentrations and pH in hind limb muscle of WT andMAK�/� miceAll values are presented as mean � S.D. Muscle tissue concentrations of Pi and PCrare calculated assuming a tissue �ATP of 7.8 mM and corrected for T1 relaxationeffects.

�PCr �Pi pH

mM mM

WT 23.7 � 2.3 2.8 � 0.4 7.27 � 0.08MAK�/� 21.0 � 2.6a 2.6 � 0.8 7.25 � 0.04

a Statistical difference determined with a two-tailed student t-test (p 0.05).

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NOEs could be detected. The different behavior of ATP in vivoand in vitro becomes clear when we consider the relaxationproperties of the phosphorous spins in ATP in more detail. Ithas beenwell established that two relaxationmechanisms dom-inate the relaxation behavior of the 31P spins inATP: the dipole-dipole relaxation and the relaxation induced by the chemicalshift anisotropy (CSA). For a 31P two-spin system, e.g. the �-and �-phosphates in ATP, the longitudinal relaxation is deter-mined by the auto-relaxation constants �dip and �CSA arisingfrom the dipole-dipole and the CSA relaxation, respectively,and by the cross-relaxation constant,�, arising from the dipole-dipole interaction. The contributions of these different relax-ation constants to the relaxation behavior of a two-spin 31P-31Pspin system with an interspin distance of 3 Å have been plottedin Fig. 5A as a function of the rotational correlation time, c.This figure shows that the CSA contribution is dominating therelaxation entirely for 3 � 10�11 c 2 � 10�8 s. For largertumbling times, i.e. c � 2 � 10�8 s, the dipolar relaxation startsto contribute significantly, and for c � 10�7 s, it becomes thepredominant relaxation mechanism. For ATP free in solution,c � 0.3 ns (22) (indicated in Fig. 5 by the dashed vertical lines).As expected, in this situation, the CSA relaxation rate constant(�CSA � 0.718 s�1) largely exceeds the auto-relaxation andcross-relaxation constants due to dipolar interactions, whichamount to �dip � 0.006 s�1 and � � 0.003 s�1. Given theexpression for the steady state NOE (for derivation, see supple-mental material),

ss �MzA

� � MzA0

MzA0 �

�

�dip � �CSA(Eq. 2)

we expect a negligible steady state NOE effect (ss � �0.004).Only when the dipole-dipole mechanism becomes predomi-nantmayNOEs be induced. This is shown in Fig. 5B (solid line),where the steady state NOE has been plotted as a function of c.It is clear thatNOEeffects are negligible formoleculeswith c10 ns, which is in agreement with the aforementioned experi-ments on ATP free in solution.Transferred NOE in ATP—Further examination of the solid

curve in Fig. 5B shows thatNOE effects can only be expected forhigher values of the rotational correlation time, e.g. the NOEeffects observed in vivo for the phosphates inATP (ss ��0.25)occur when c � 30 10�9 s. This would mean that c hasincreased by a factor of 100, when considering in vivo ratherthan in vitro conditions. This seems very unlikely; severalexperiments indicate that the c of cytosolic ATP in skeletalmuscle does not deviate much from its in vitro value (see “Dis-cussion”). However, the effective correlation time of ATPincreases when its rotational motion is restricted by binding tolarger molecules. In case exchange between the bound and freeATP occurs, NOE effects generated in the bound state may betransferred to ATP in the free state, designated the transferredNOE (trNOE). For this situation, the expression for the steadystate trNOE is given by the equation below (see supplementalmaterial and Ref. 23).

ss ���f � kfb � kbf��b

��b � kbf���f � kfb)�kfbkbfPb �

��b � kfb � kbf��f

��b � kbf���f � kfb� � kfbkbfPf

(Eq. 3)

Here, kfb is pseudo first order association rate constant ofcomplex formation, kbf is the dissociation rate constant, Pband Pf are the fractions of ATP in the bound and free state,respectively, �f and �b are the auto relaxation rate constantsin the free and bound state, and similarly, �f and �b are thecross-relaxation constants in the free and bound state. UsingEquation 3, the steady state trNOE effect was calculated as afunction of c of the bound fraction (c,bound), for Pb equal to5 and 10% (Fig. 5B, dashed lines). In the calculation it wasassumed that kfb � kassoc[E] in which kassoc � 1 � 108 M�1s�1

(23), the association rate constant, and kbf � 9000 s�1, lead-ing to a concentration of free binding sites [E] � 1.10�5 M.Examination of Fig. 5B shows that ATP has to bind to struc-tures with a c,bound � 10�6 s to be able to account for theobserved trNOEs. The result is not very sensitive to thechoice of kbf (see below and Fig. 5D).Combining Transferred NOE and ATP-ADP Exchange—

Above, we have shown that theMT effects between ATP phos-phate signals arise from cross-relaxation, which is responsiblefor the observed NOEs (see supplemental Fig. S1). However, itseems odd that the decrease of the �-ATP signal during selec-tive saturation of the �-ATP/�-ADP resonance is not influ-enced by ATP-producing chemical exchange reactions. Toinvestigate this aspect, we considered the following reaction.

FIGURE 3. The 31P MR signal of PCr under the influence of the saturationof the �-ATP resonance as a function of the duration of the saturatingpulse. The length of the saturation pulse, from left to right, is: 0.2, 0.5, 1, 1.5, 2,3, and 5 s. PCr signals are shown for MAK�/� (left) and WT mice (right). Linebroadening is 5 Hz.

TABLE 2Unidirectional pseudo first order rates and fluxes in muscle of WTand MAK�/� miceRates and fluxes were determined from decreases in PCr and Pi upon saturation ofthe �-ATP/�-ADP resonances. All values are presented as mean � S.D.

WT MAK�/�

PCr3 ATPkCK,for (s�1)a 0.43 � 0.05 0.04 � 0.01b�PCr (s�1) 0.41 � 0.06FCK,for (mM � s�1) 10.2 � 1.5 0.84 � 0.24b

Pi3 ATPkPi,for (s�1) 0.23 � 0.03c 0.18 � 0.04FPi,for (mM � s�1) 0.64 � 0.13 0.47 � 0.18

a To improve the accuracy of the determination of the small kCK,for in the MAK�/�

mice the value for T1int was assumed to be equal to the value determined in the

WT mice and for both MAK andWT.b Curve is significantly different fromWT (F-test using a 0.95 confidence interval).c The mono exponential function for Pi3 ATP was fitted according to Equation 1assuming an intrinsic auto relaxation time of 1.95 s (19, 39).

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ATPbound -|0kbf

kfb

ATPfree 9|=kADP,for

kADP,rev

ADP

REACTION 3

The left-hand part of this expression represents the exchangebetween bound and free ATP, and the right-hand part repre-sents the transition between �-ATP and �-ADP mediated byCK, AK, ATPases, and/or GAPDH/phosphoglycerate kinaseactivities. We focus on the behavior of the �-ATP resonancewhen the �-ATP and �-ADP resonances are saturated. Thesteady state MT effect for this reaction scheme is presented inEquation 4 (for the derivation, see supplemental material). Thefirst two terms on the right-hand side give rise to the trNOE,discussed in the previous section; the last term on the right-hand side represents the effect of the additional exchange onthe �-ATP resonance.

ss ��kbf � �f � kADP,rev � kfb��b

���b � kbf����f � kADP,rev � kfb� � kfbkbfPb

��kfb � �b � kbf��f

���b � kbf����f � kADP,rev � kfb� � kfbkbfPf

����b � kbf � kfb�kADP,rev

���b � kbf����f � kADP,rev � kfb� � kfbkbfPf (Eq. 4)

The pseudo first order rate constant of the reverse CK reac-tion was determined inWTmice by saturating the PCr spins:kCK,rev � 1.4 s�1. It dominates over the contribution of theATPase/glycolysis in the ATP3 ADP reaction in resting skel-etal muscle (�0.08 s�1) and the AK reaction, which is known tobe only 15% of the total ATP turnover inWT skeletal muscle atrest (18). Using Equation 4, we calculated the MT effectexpected on the �-ATP resonance for kADP,rev ranging from0.01 to 1.4 s�1 (Fig. 5C). For the maximum value (1.4 s�1),correspondingwith theCK-mediated exchange, the�-ATP sig-nal shows a reduction of 65%. Thus far, such a large MT effecton the �-ATP signal has not been detected in vivo.To assess the effect of different conversion rates between free

and bound ATP on theMT effect on the �-ATP signal, we calcu-

lated this effect, including ADP-ATPexchange,withkADP,rev� 1.4 s�1, fordifferent values of kfb and kbf (Fig.5D). This reveals that the influenceof these rates is negligible.

DISCUSSION

The participation of ATP inphosphoryl exchange reactions canbe analyzed by MT experiments in31PMRspectroscopy.However,MTmay also occur by cross-relaxationbetween spins leading to signalattenuation called NOE. The natureand extent of NOEs can provideinteresting information on the bio-physical state of molecules in cells.The MT effect on the �-ATP reso-

nance after saturating the co-resonating �-ATP and �-ADPsignals is of particular interest as it may arise both from cross-relaxation and from chemical exchange, which in muscles isdominated by cytosolic muscle CK and AK reactions. Toresolve this ambiguity, we performed MT experiments onskeletal muscles of wild type mice and of mice lacking CKand AK activity. The results indicate that in resting muscles,a small fraction of ATP is bound to large macromolecules,and that the magnetization effect on the �-ATP resonancementioned above is caused by cross-relaxation.Cross-relaxation between the Phosphorous Spins in ATP—

MT experiments carried out on solutions of ATP do not dem-onstrate the presence of 31P-31PNOEs (see “Results” andRefs. 5and 24). Here, however, we present several experiments thatshow that Overhauser effects between the phosphorous spinsof ATP do occur in vivo. A case in point is the experiment inwhich saturation of the �-ATP signal leads to decreases in thePCr signal in WT mice. Because the �-ATP phosphate is notconverted into the phosphate group of PCr by any reaction, thisdecrease can only be attributed to a relayed Overhauser effect(25), i.e. magnetization is transferred by cross-relaxation from�-ATP to �-ATP and subsequently to the PCr signal throughthe CK reaction. In MAK�/� mice, the flux through the CKreaction is lowered so much that the last step becomes ineffec-tive, explaining why the effect is no longer observed (Fig. 3).By the same token, saturation of the �-ATP resonance leads

to a reduction of the �-ATP resonance intensity. Again,because no direct or relayed reactions are known in which an�-phosphate is promoted to a �-phosphate in ATP, the reduc-tion in signal intensity has to be attributed to an NOE effect.Saturation of the�-ATP resonance not only leads to an effect

on the PCr signal but also reduces the intensity of the�- and the�-ATP resonances, and it does so in a symmetric way, i.e. thesignal reductions are about the same as that of the �-ATP res-onance if either the �-ATP or the �-ATP resonances are satu-rated. This is expectedwhen dipole-dipole relaxation is govern-ing theMT. These physical considerations are corroborated bythe results obtained for the double-mutantMAK�/� mice. Themutants, which are M-CK-deficient, exhibited only a minorflux through the (residual mitochondrial) CK reaction in both

FIGURE 4. Saturation transfer effects between 31P spins within ATP as a function of the duration of thesaturation pulse. MT effects between �-� and �-� are similar for both WT and MAK�/� mice. MT effectsbetween �- and �-ATP were small and within the detection limits (see results obtained for WT mice) Signalintensities are presented as mean values � S.E. Dotted lines represent fits of monoexponential curves to theexperimental data assuming � to be equal in all mice and for all ATP signals.

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directions. As theAK activity inMAK�/�mice is only about 1%when compared with WT mice (18), we assumed negligiblefluxes through the AK reaction as well. In this light, the equalreductions of the �-ATP signal in WT and MAK�/� mice leadus to conclude thatM-CK andAK1 activity contribute insignif-icantly to the observedMTeffect. Furthermore, it is remarkablethat not only are all steady state MT effects between nearestneighbor phosphates about the same inWTandMAK�/�micebut also that the time constants of the signal decays are equal.Transferred NOEs—Although the preceding section implies

thatNOEeffects explain theMTbetweenATPphosphates seenin vivo, we have to discuss the observation that no NOEs areseen for solutions of ATP. Overhauser effects are only observedwhen the dipole-dipole relaxation mechanism dominates thespin-lattice relaxation. It is, however, well established that 31Pspins are also subjected to relaxation induced by the chemical

shift anisotropy (26, 27). We calculated the auto-relaxationconstants, �dip and �CSA, and the cross-relaxation constant, �,for a 31P two-spin system as a function of c and demonstrated,given that c �3 � 10�10 s for ATP in solution, the CSA contri-bution dominates the relaxation process (Fig. 5A). Conse-quently, the NOE effect in ATP in solution is negligible (Fig.5B). It is generally assumed that the rotational correlation timeof cytosolic ATP is not too different from that of ATP in solu-tion. This is reasonable because the translational diffusion ofcytosolic ATP is only reduced by a factor of 2–3 (28–30), andthe c of small proteins in intact oocytes diminishes by the sameamount when compared with that in aqueous solutions (31).This means that the observed NOEs can only be interpreted asbeing transferred NOEs. When the rotational motion of ATPbecomes restricted by binding to larger molecules, this is feasi-ble. Given that there exists a dynamic equilibrium between the

FIGURE 5. Spin-lattice relaxation rate constants, steady state NOEs, and trNOEs as a function of �c derived for a 31P two-spin system. A, the differentrelaxation rate constants contributing to the relaxation in a 31P-31P system. �dip and �CSA are the auto-relaxation rate constants due to dipole-dipole interac-tions (gray) and chemical shift anisotropy (CSA, black), and � is the cross-relaxation rate constant (gray, dashed). B, steady state Overhauser enhancement factor(ss) for a 31P-31P two-spin system (gray) as a function of c calculated on the basis of Equation 2 and steady state transferred NOE as a function of the c of thebound fraction (c,bound) calculated using Equation 3 for Pb � 10% and Pb � 5% (dashed line). C, simulated steady state MT effects on �-ATP due to trNOE andATP3ADP conversion based on Equation 7. trNOE without (dark dashed line) and with ATP3ADP conversion assuming kADP,rev � 0.01 s�1, kADP,rev � 0.1 s�1,kADP,rev � 1 s�1, and kADP,rev � 1.4 s�1 (solid lines). Note that for kADP,rev � 1.4 s�1 (corresponding with the reverse rate of the CK reaction), a reduction of about65% is expected at the �-ATP signal. D, simulated steady state MT effect due to transferred NOE for varying exchange rates between the free and bound ATPpools: kfb/kbf � 1000 s�1/9000 s�1 (solid line), kfb/kbf � 10 s�1/90 s�1 (dashed line), and kfb/kbf � 1 s�1/9 s�1 (dotted line), where kADP,rev � 1.4 s�1. Note that kfband kbf have a minor influence on ss only for c,bound �� 10�6. All plots were calculated based on a 31P resonance frequency of 121.4 MHz and a distancebetween the phosphorous nuclei r � 3.0 � 10�10 m.

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free and a “bound” form of ATP, NOEs created in the boundform may be transferred to the free form, where they becomeNMR visible as trNOE effects.To estimate the magnitude of these effects, we calculated

trNOEs assuming that 5 or 10% of the intracellular ATP occursin the bound form. Although an enormous variety of proteinsare known to form complexes with ATP (32), the calculationsshow that we can already rule out a large class of proteins; the cof theATP complexesmust be around 10�6 s to obtain a trNOEof about 20% for the�-ATP signalwhen the�-ATP resonance issaturated (Fig. 5B). This sets a limitation to the size ofmolecularstructures involved. Smaller enzymes such as CK (81-kDadimeric isoform with a c of 7.4 ns (22)) and/or AK (25-kDamonomeric isoform1) can thus be disregarded as candidates. Infact, this is confirmed by the similar NOEs observed for theMAK�/� and WT littermates.

Not only does the size of a receptor molecule determine themagnitude of the trNOE.The rotationalmotion of a smallATP-binding protein can also be greatly restricted when it is embed-ded in a membrane or cytoskeleton-bound. This was, forinstance, demonstrated for adenosine nucleotide translocases(�30 kDa), which, when embedded in the inner mitochondrialmembrane, obtain a c � 10�6 s (33). The binding of ATP tomacromolecules does not necessarily need to be functional inenergy metabolism. The adenosine moiety and phosphategroup make ATP a suitable candidate for stacking and electro-static interactions with other compounds (32).Absence of Exchange-mediated MT Effects on the �-ATP

Signal—The conclusion that the reduction of the �-ATP signalupon saturation of the �-ATP signal is a trNOE effect raises thequestionwhy chemical�-ATP3�-ADP conversions do not oronly negligibly contribute to this effect? To investigate this, wederived an expression for the steady state reduction of the�-ATP signal (Equation 4) for the situation that both trNOEand ADP-ATP exchange are present. For simplicity, we onlyincorporated the MT effect arising from the reverse CK reac-tion (i.e. kADP,rev � 1.4 s�1, Table 2). This leads to an expectedreduction of 65% of the �-ATP signal, which is at odds with ourexperimental results and those reported in the literature, wherereductions of 20–30% have been reported. It is noted in passingthat a similar observation has been made previously by LeRumeur et al. (5) in their study of rat skeletal muscle. Account-ing for additional phosphorylation reactions (i.e.AK, glycolyticactivity, ATPases, and nucleotide di- or monophosphate ki-nases) would have lowered the expected �-ATP resonanceintensity even more.The calculation of the exchange-mediated reduction of the

�-ATP signal is based on accepted practice to use the creatinekinase equilibrium to derive the free cytosolic ADP concentra-tion. The fact that in vivo no MT effects �0.65 have been seenon the �-ATP resonance casts serious doubts on this approach.

However, relatively large MT effects can be observed in invitro experiments, as reported by Koretsky et al. (24) and Brin-dle and Radda (34). They showed that for solutions containingCK, PCr, Cr, ADP, ATP, and free Mg2� mimicking the in vivoconcentrations presumed for the near equilibriumCK reaction,saturation of the �-ATP/�-ADP indeed resulted in a significantreduction of the �-ATP resonance intensity. The effect could

not be attributed to an NOE effect, which accords with ouranalysis; NOEs cannot be expected under in vitro conditions.Thus, in this situation, the reduction of the �-ATP signal iscaused by the ATP to ADP conversion.So how can the apparently conflicting in vivo and in vitro

results, of which the latter are in accordance with the predic-tions in Fig. 5C, be reconciled? The following possibilities cometo mind. 1) The �-ADP signal is not properly saturated. 2) The�-ADP is properly saturated, but the effect of the �-phosphorylexchange is outcompeted by spin-lattice relaxation due to ATPbinding to various proteins or receptors. 3) The ADP takingpart in the CK reaction is in a state in which its �-resonancecannot be saturated.With respect to the first point, we notice that the co-satura-

tion of the �-ADP resonance (�0.4 ppm upfield from �-ATP)may not be optimal. However, control experiments inwhichweobserved an unchanged signal reduction of the �-ATP inhuman muscle at 3 tesla, when the carrier frequency of thesaturation pulsewas varied from�0.1 to�0.8 ppm (steps 0.05–0.1 ppm) and the observations of Koretsky et al. (24) render thisoption very unlikely.In situation 2, �-ADP is saturated, but due to its low concen-

tration with respect to ATP, the effect of �-phosphorylexchange is less effective than that of exchange between �-ATPand PCr. Basically, this need not form an impediment for theobservation of the �-phosphoryl exchange as was shown by invitro experiments (24, 34). However, in vivo, ATP is involved inmany reactions in which it binds to proteins, and one couldimagine that the effect of the �-phosphoryl exchange on the�-ATP resonance is undone by amuchmore effective T1 relax-ation than in the in vitro situation. Careful examination of Fig.5A shows that this is not to be expected. At in vitro-like condi-tions (c �0.3 ns), the T1 relaxation through the chemical shiftanisotropy is very effective, and still, the MT effect of �-phos-phoryl exchange is observed (24, 34). For higher values of therotational correlation times, corresponding with ATP bindingto medium-sized proteins, T1 becomes even longer. For com-plex formation, leading to much longer rotational correlationtimes (e.g. 10�6 s), T1 becomes shorter, but not dramatically(T1 � 0.5 s). In Equation 7, which describes the decrease of the�-ATP resonance as a result of �-phosphoryl exchange and thebinding of ATP to slowly tumbling receptors at the presumedCK equilibrium, these effects are incorporated, and it leads tothe reduction of the �-ATP signal indicated in Fig. 5C. In sum-mary, at the conditions prevailing in our experiments, thisrelaxation effect does not nullify the MT effect of the �-phos-phoryl exchange.In situations 1 and 2, the assumption that the cytosolic ADP

concentration can be derived from the CK equilibrium stillholds. This is no longer true for situation 3, which we considerbelow. Here, we deal with ADP that is involved in the CK reac-tion, whereas its �-resonance cannot be saturated.A Model for ADP 7 ATP Conversion—For situation 3, we

propose a model in which the CK reaction proceeds via tran-siently free ADP, which is drawn from a pool of bound ADP ofwhich the spin population cannot be saturated (Fig. 6). Thissituation can be represented by the following two equations

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ADPbound -|0kst

kts

ADPtrans

REACTION 4

and

ADPtrans � CK -|0kADP_CK,for

kADP_CK,rev

ATP � CK

REACTION 5

Here, ADPbound is the bound, non-saturableADP, andADPtransis the ADP transiently present in the cytosol. For this situation,the overall rate constants for the conversion of ADPbound toATP (kex) and its reverse (kex,rev) are

kex �kstkADP_CK,for�CK

kts � kADP_CK,for�CK(Eq. 5)

and

kex,rev �ktskADP_CK,rev�CK

kts � kADP_CK,for�CK(Eq. 6)

In this model, the steady state situation is maintained, and anequilibrated free concentration of ADP, commonly derivedfrom the CK equilibrium, need not be invoked.

We consider the limit that kts �� kADP_CK,for[CK]. In thiscase, the overall rate constants reduce to

kex �kst

ktskADP_CK,for�CK (Eq. 7)

and

kex,rev � kADP_CK,rev�CK (Eq. 8)

Characteristic of this limit is that ADP exchanges manytimes between the free and the bound state before it getscaught in the CK reaction. In other words, the overall for-ward reaction (kex) proceeds via a pre-equilibrium, whichlies far to the left. In the reverse direction, the reaction fromATP to ADPtrans, kADP_CK,rev, and the CK concentration deter-mines the overall rate constant. Thus, in both directions, theoverall rate constants are linearly dependent on [CK]. This con-curs with the observation that the pseudo first order rate con-stants (kCK,for and kCK,rev) that can be derived fromFig. 2 for themutant andWTmice indicate that they are proportional to CKactivity, reflecting its concentration. A linear relation betweenkCK,for and CK activity (Vmax) has been noticed previously withdifferent expression levels of the M-CK isoform (35).It is inherent in the present approach that saturation of the

spin population of bound ADP is not possible. This conditionapplies if the ADP resonance is inhomogeneously broadened,which occurs, for example, when ADP is bound to relativelyrigid cellular structures with a solid state-like character. Due tothe anisotropy of the chemical shift, the resonances are thenspread over a region of about 180 ppm, and only a negligiblepart of the bound spin population will be saturated. Relativelyrigid cellular structures with ADP binding capacity, e.g. compo-nents of the cell cytoskeleton, for instance, myofibrils and actinfilaments, are possible candidates for this type of binding (36),but mitochondria have also been suggested for this role (2).Conclusions—Central to the discussion in this study has been

the small reduction (�20%) of the �-ATP signal upon satura-tion of the �-ATP signal. Understanding this effect requiresthat a distinction is being made between chemical exchangeand cross-relaxation and that the relaxation properties of thephosphorous spins are properly accounted for. The resultsobtained show that the reduction of the �-ATP signal is causedby transferred NOE effects made possible by binding-unbind-ing of ATP, i.e. exchange between a state of free diffusion andbound state in which the rotational freedom is restricted. Thereduction of the �-ATP signal is not or only negligibly causedby �-phosphoryl exchange. Consequently, ADP participatingin the CK reaction comes from a source where its spin systemcannot be saturated. To account for these results, we propose amodel in which free cytosolic ADP is only transiently present inskeletal muscle at rest. The concentration of this ADP is muchless than that of the predicted free [ADP], derived by calcula-tion from the CK equilibrium, which is already too low for invivo detection by 31P MR spectroscopy.

It remains to be investigated whether in circumstances ofhigh intensity exercise in muscle, the free ADP concentrationmay increase to such an extent that its binding sites on the solidstate-like structures become saturated and the free ADP pool

FIGURE 6. Scheme of the phosphoryl exchange reactions in which cytoso-lic ATP is involved, including the role of bound ATP and ADP. The NOEsgenerated in the bound ATP are transferred to the free ATP through theexchange reaction characterized by kst and kts. The ATP7 ADP conversionproceeds via transiently free ADP. The rate constant kex,rev does not representa separate path but is a combination of the rate constants in the individualreaction steps (see Equations 5 and 6). The scheme presented explains theexperimental observations.

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might become accessible to NMR saturation. To our knowl-edge, NMR visible in vivo signals of free ADP have only beenobserved in AK1-deficient muscle during isometric tetaniccontractions (37).The reduction of the �-ATP signal upon saturation of the

�-ATP resonance has been reported in numerous studies. Forinstance, in an early study on perfused heart (14) and later,similar effects have been reported repeatedly for hearts (7, 12,13), as well as for skeletal muscle (5, 10, 11), for kidney (7), forT47D human breast cells (9), for Chlamydomonas reinhardtii(38), and for rat and human brain (6, 8). Taken collectively,these findings imply that the observed magnetization transferbetween �-ATP and �-ATP is not tissue-specific. Interestingly,the similarity of the effect suggests a common mechanism, i.e.mode of ATP binding in different cell types and tissues. Untilnow, the MT effect was frequently attributed to ATP7 ADPexchange mediated by CK, AK, and/or ATPases. In view of thepresent results, it is more likely due to transferred NOE effects.

Acknowledgments—We thank F. Oerlemans for help in the labora-tory, A. Veltien for assistance with the NMR experiments, T. Scheenenfor implementing the saturation transfer sequence on the 3 tesla-scan-ner, and H. E. Kan for helpful discussions.

REFERENCES1. Ellington, W. R. (2001) Annu. Rev. Physiol. 63, 289–3252. Alger, J. R., and Shulman, R. G. (1984) Q. Rev. Biophys. 17, 83–1243. Brindle, K. M. (1988) Prog. Nucl. Magn. Reason. Spectrosc. 20, 257–2934. Campbell, S. L., Jones, K. A., and Shulman, R. G. (1985) FEBS Lett. 193,

189–1935. Le Rumeur, E., Le Tallec, N., Kernec, F., and de Certaines, J. D. (1997)

NMR Biomed. 10, 67–726. Du, F., and Chen, W. (2008) in Proceedings of the International Society of

Magnetic Resonance in Medicine, 16th Scientific Meeting and Exhibition,Toronto, May 3–9, 2008, p. 2337, International Society for Magnetic Res-onance in Medicine, Berkeley, California

7. Koretsky, A. P., Wang, S., Klein, M. P., James, T. L., and Weiner, M. W.(1986) Biochemistry 25, 77–84

8. Shoubridge, E. A., Briggs, R. W., and Radda, G. K. (1982) FEBS Lett. 140,289–292

9. Neeman, M., Rushkin, E., Kaye, A. M., and Degani, H. (1987) Biochim.Biophys. Acta 930, 179–192

10. Ravalec, X., Le Tallec, N., Carre, F., de Certaines, J. D., and Le Rumeur, E.(1999)Magma 9, 52–58

11. Shoubridge, E. A., Bland, J. L., and Radda, G. K. (1984) Biochim. Biophys.Acta 805, 72–78

12. Ugurbil, K., Kingsley-Hickman, P. B., Sako, E. Y., Zimmer, S., Mo-

hanakrishnan, P., Robitaille, P. M., Thoma, W. J., Johnson, A., Foker, J. E.,and From, A. H. (1987) Ann. N.Y. Acad. Sci. 508, 265–286

13. Goudemant, J. F., vander Elst, L., Dupont, B., Van Haverbeke, Y., andMuller, R. N. (1994) NMR Biomed. 7, 101–110

14. Matthews, P. M., Bland, J. L., Gadian, D. G., and Radda, G. K. (1981)Biochem. Biophys. Res. Commun. 103, 1052–1059

15. McGilvery, R.W., andMurray, T.W. (1974) J. Biol. Chem.249, 5845–585016. Wiseman, R. W., and Kushmerick, M. J. (1995) J. Biol. Chem. 270,

12428–1243817. Veech, R. L., Lawson, J. W., Cornell, N.W., and Krebs, H. A. (1979) J. Biol.

Chem. 254, 6538–654718. Janssen, E., Terzic, A., Wieringa, B., and Dzeja, P. P. (2003) J. Biol. Chem.

278, 30441–3044919. in ’t Zandt, H. J., de Groof, A. J., Renema, W. K., Oerlemans, F. T., Klomp,

D. W., Wieringa, B., and Heerschap, A. (2003) J. Physiol. 548, 847–85820. Moon, R. B., and Richards, J. H. (1973) J. Biol. Chem. 248, 7276–727821. Bachert, P., and Bellemann, M. E. (1992) J. Magn. Res. 100, 146–15622. Murali, N., Jarori, G. K., Landy, S. B., and Rao, B. D. (1993) Biochemistry

32, 12941–1294823. Clore, G., and Gronenborn, A. (1982) J. Magn. Reason. 48, 402–41724. Koretsky, A. P., Basus, V. J., James, T. L., Klein, M. P., and Weiner, M. W.

(1985)Magn. Reson. Med. 2, 586–59425. Van de Ven, F. J., Janssen, H. G., Graslund, A., and Hilbers, C.W. (1988) J.

Magn. Reason. 79, 221–23526. Brauer, M., and Sykes, B. D. (1981) Biochemistry 20, 2060–206427. Mathur-De Vre, R., Maerschalk, C., and Delporte, C. (1990)Magn. Reson.

Imaging 8, 691–69828. de Graaf, R. A., van Kranenburg, A., and Nicolay, K. (2000) Biophys. J. 78,

1657–166429. Sehy, J. V., Ackerman, J. J., and Neil, J. J. (2002) Magn. Reson. Med. 48,

42–5130. Vendelin, M., and Birkedal, R. (2008) Am. J. Physiol. Cell Physiol. 295,

C1302–C131531. Wagner, B., Tharmann, R., Haase, I., Fischer, M., and Bausch, A. R. (2006)

Proc. Natl. Acad. Sci. U.S.A. 103, 13974–1397832. Mao, L., Wang, Y., Liu, Y., and Hu, X. (2004) J. Mol. Biol. 336, 787–80733. Horvath, L. I., Munding, A., Beyer, K., Klingenberg, M., and Marsh, D.

(1989) Biochemistry 28, 407–41434. Brindle, K. M., and Radda, G. K. (1985) Biochim. Biophys. Acta 829,

188–20135. Askenasy, N., and Koretsky, A. P. (2002) Am. J. Physiol. Cell Physiol. 282,

C338–C34636. Martonosi, A., Gouvea, M. A., and Gergerly, J. (1960) J. Biol. Chem. 235,

1700–170337. Hancock, C. R., Brault, J. J., Wiseman, R. W., Terjung, R. L., and Meyer,

R. A. (2005) Am. J. Physiol. Cell Physiol. 288, C1298–130438. Hentrich, S., Hebeler, M., Grimme, L. H., Leibfritz, D., and Mayer, A.

(1993) Eur. Biophys. J. 22, 31–3939. Cline, G.W., Vidal-Puig, A. J., Dufour, S., Cadman, K. S., Lowell, B. B., and

Shulman, G. I. (2001) J. Biol. Chem. 276, 20240–20244

Differential Intracellular Binding of ATP and ADP

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HeerschapChristine Nabuurs, Bertolt Huijbregts, Bé Wieringa, Cees W. Hilbers and Arend

Macromolecular Association of ATP and ADP in Skeletal MuscleP Saturation Transfer Spectroscopy Predicts Differential Intracellular31

doi: 10.1074/jbc.M110.164665 originally published online September 29, 20102010, 285:39588-39596.J. Biol. Chem. 

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