©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Adenylate Kinase-catalyzed Phosphoryl Transfer Couples ATP Utilization with Its Generation by Glycolysis in Intact Muscle (*)

(Received for publication, November 2, 1994; and in revised form, January 9, 1995)

Robert J. Zeleznikar Petras P. Dzeja Nelson D. Goldberg (§)

From the Department of Biochemistry, University of Minnesota, Medical School, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We previously suggested that an importance of adenylate kinase (AdK) in skeletal muscle is to function as a high energy phosphoryl transfer system regulating ATP generation in correspondence with its consumption by specific cellular processes. The present experiments are intended to define the ATP-generating system coupled to and regulated by AdK-catalyzed phosphotransfer in skeletal muscle and also to examine the relationship between AdK- and creatine kinase (CK)-catalyzed phosphotransfer. Rates of phosphoryl transfer catalyzed by AdK were assessed in intact, isolated rat diaphragm by determining rates of AMP phosphorylation with endogenously generated [-^18O]ATP under conditions of altered anaerobic and aerobic ATP production. AdK-catalyzed phosphoryl transfer rates accelerated incrementally up to 12-fold in direct proportion to stimulated contractile frequency in parallel with equivalent increases in rates of ATP generation by lactate producing glycolysis. Stoichiometric equivalent increases of AdK-catalyzed phosphotransfer and anaerobic ATP production also occurred up to more than 20-fold when oxidative phosphorylation was impaired by either O(2) deprivation or treatment with KCN or p-(trifluoromethoxy)-phenylhydrazone. These enhanced rates of AMP phosphorylation were balanced by virtually identically increased rates of AdK-catalyzed generation of AMP. This AMP was traced to arise from AdK-catalyzed phosphotransfer involving ADP generated by a muscle ATPase. Increased AdK-catalyzed phosphotransfer paired with the apparent compensatory increase in ATP generation by anaerobic glycolysis in oxygen-deprived muscle occurred coincident with diminished rates of CK-catalyzed phosphoryl transfer indicative of a pairing between oxidatively produced ATP and CK-catalyzed phosphotransfer. A metabolic model consistent with these results and conforming to the Mitchell general principle of vectorial ligand conduction is suggested.


INTRODUCTION

We have recently obtained the first information about the kinetics and subcellular compartmental properties of AdK (^1)in the intact muscle cell(1, 2) . The characteristics of AdK behavior in the intact cell provides a much different perspective from which to define its biological importance than has been possible from several decades of assessing its catalytic properties by conventional cell-free analyses(3) . In the intact muscle cells comprising the rat diaphragm, the net AdK-catalyzed transfer of phosphoryls was found to be restricted to a limited number of discrete subcellular compartments that increase in size and/or number with increasing frequency of contractions(1) . This increased rate of AdK-catalyzed phosphotransfer, associated with muscle contraction, occurred with minimal or no measurable change in the steady state levels of AMP, ADP, and ATP. The greatly restricted velocity of AdK in the intact muscle compared to its much greater activity measurable by cell-free analysis indicates that AdK-catalyzed phosphotransfer is not merely involved in equilibrating cellular adenine nucleotides according to the traditional view of its metabolic importance. If contraction-associated bursts of AdK-catalyzed phosphotransfer were only for this purpose, increases in AMP concentration should have accompanied each contraction and most likely would have been detected following the almost 1000 contractions that occur during 4 min of contraction at 4 Hz. Instead of an accumulation of AMP, the most prominent metabolic characteristic of AdK-catalyzed phosphotransfer associated with contraction was the apparently very rapid phosphorylation of AMP. To measure rates of AdK-catalyzed phosphorylation of AMP in intact muscle, a procedure of ^18O-phosphoryl labeling was used that detects only net AMP phosphorylation by anabolically generated ATP; it does not detect reversals of AdK catalysis whereby ATP formed in the forward reaction serves as the phosphoryl donor in the reverse reaction. Therefore, this previously undetected AMP metabolic flux appeared to represent a functional link with characteristic stoichiometry between contractile and metabolic processes.

These results suggested a model of metabolic regulation (1) that provides a function for AdK, similar to that suggested by Bessman and Carpenter (4) and also by Dzeja et al. (5), of transferring high energy phosphoryls from a metabolic process generating ATP to cellular components consuming ATP in functionally definable subcellular locales. Also suggested from preliminary observations not documented experimentally in our previous report (1) was that in rat diaphragm muscle the source of the newly synthesized ATP involved in the AdK-catalyzed phosphotransfer derives from glycolysis resulting in lactate formation.

The results described here provide experimental evidence that in rat diaphragm a major function of AdK is to catalyze the transfer of high energy phosphoryls generated by anaerobic glycolysis in close correspondence with ADP produced by a contraction-related event. They also show an interrelationship between AdK- and creatine kinase (CK)-catalyzed phosphotransfer which is manifest as reciprocal changes in rate when oxidative ATP production is impaired. Oxygen deprivation resulted in a decrease in phosphotransfer catalyzed by CK and a shift to greater rates of phosphotransfer catalyzed by AdK paralleling compensatory increases in anaerobic glycolytic flux. A model of metabolic regulation consistent with these experimental results is presented whereby AdK-catalyzed transfer of nucleotide phosphoryls can rapidly regulate and coordinate the rate of ATP generation by anaerobic glycolysis with the rate of ATP utilization by specific cellular events supported by ATP deriving from this glycolytic process. It also shows an interrelated coupling of the AdK and CK phosphotransfer systems to anaerobic glycolytic and oxidative ATP production, respectively.


EXPERIMENTAL PROCEDURES

Tissue Preparation and Incubation

Intact rat diaphragms were obtained (6) from male Sprague-Dawley rats (Bio-Lab, 150-200 g) and fed ad libitum. The procedure for incubating and stimulating this tissue has been described in detail(1) . Briefly, incubation in medium containing [^18O]water (Monsanto Co. and Isotec; 25-40% enriched) was initiated by rapidly transferring the tissue, ligated to a Lucite holder, to 12 ml of physiological buffer (1) continuously bubbled with O(2)/CO(2) (95:5%) except when N(2)/CO(2) (95:5%) was used to equilibrate the medium with nitrogen. Incubations were terminated by freezing with Wollenberger clamps cooled with liquid N(2) or by plunging the tissue into liquid N(2). The muscle was dissected free of bone and connective tissue while maintained below -20 °C, then ground to a fine powder in liquid nitrogen, and extracted with 3 M perchloric acid at -10 °C. Details of the purification and enzymic preparation of high energy phosphoryls for analysis have been described(1, 7) . In some experiments when inhibitors of oxidative phosphorylation were tested, diaphragms were carefully dissected free of the ribs before incubation in [^18O]water.

Analysis of [^18O]Phosphoryl Labeling

Analysis of the ^18O enrichment of phosphoryls of -ATP, beta-ADP, beta-ATP, and creatine phosphate (CrP) was accomplished using gas chromatography-mass spectrometry to monitor mass ions of 357, 359, 361, and 363 corresponding to ^18O(0), ^18O(1), ^18O(2), and ^18O(3) phosphoryl species, respectively, following enzymic transfer of the nucleotide phosphoryls to glycerol forming glycerol 3-phosphate and its conversion to the trimethylsilyl derivative(2, 7) . The rate of AdK catalysis in the intact muscle cell was determined from the rate of appearance of ^18O-containing phosphoryls in the beta-position of ADP and ATP. These phosphoryls arise from the more rapid rate of ^18O appearance in the -phosphoryl of ATP which, in turn, is proportional to the rate of -ATP turnover. The in situ velocity of AdK is, therefore, a function of both the rate of AdK catalysis and the kinetics of ^18O appearance in -ATP. The -phosphoryl of ATP is transferred to AMP in the AdK-catalyzed reaction without loss or gain of ^18O atoms(7) . A computer model (Stella, High Performance Systems, Lyme NH) of AdK-catalyzed phosphoryl transfer was used to obtain estimates of the velocity of AdK-catalyzed formation of AMP from the rates of formation of [beta-^18O]ADP and [beta-^18O]ATP as described (1) . All values shown represent means ± S.E. unless otherwise designated.

Other Methods

The levels of AMP, ADP, ATP, lactate, and CrP were determined in the neutralized acid extract of muscle by coupled enzymic analysis using fluorometric detection(8) . A portion of the incubation medium was assayed to quantitate lactate release from the tissue. Protein was determined by the BCA method (Pierce). Enzymes and other chemicals were obtained from Sigma.


RESULTS AND DISCUSSION

Adenylate Kinase-catalyzed Phosphoryl Transfer in Oxygenated, Non-contracting and Contracting Muscle

In the rat diaphragm the rate of ^18O incorporation into the -phosphoryl of ATP occurs much more rapidly than ^18O-labeled beta-phosphoryls appear in ADP and ATP (1 and Fig. 2A). This marked difference in the kinetics of -ATP compared to adenine nucleotide beta-phosphoryl ^18O-labeling reveals a previously undefined, but potentially important characteristic of intact muscle cell metabolism. It shows that in the intact resting muscle cell approximately 95% of the ADP generated from hydrolytic consumption of ATP does not undergo transformation to AMP and is therefore probably not accessible to AdK. These results clearly demonstrate that AdK-catalyzed phosphotransfer must occur in highly restricted subcellular locales. This property of AdK catalysis in the intact cell is at odds with the commonly held view that the biological importance of AdK is to catalyze rapid equilibration among cytosolic adenine nucleotides(3) .


Figure 2: Kinetics of ^18O-labeled phosphoryl appearance in -ATP, beta-ATP, and beta-ADP in non-contracting rat diaphragm incubated in oxygen- or nitrogen-equilibrated medium. Diaphragm muscles were preincubated for 10 min in [O]water-containing medium that was equilibrated by continuous bubbling with 95% O(2), 5% CO(2) or 95% N(2), 5% CO(2). The tissues were then transferred to [^18O]water-containing medium equilibrated with the same gas mixture and incubated for 1, 2, or 8 min. The percentage (not computer-modeled estimates of velocities) of -ATP, beta-ATP, and beta-ADP containing 1-3 atoms of ^18O is shown. The data are corrected for small variations in the atom percent enrichment of [^18O]water in the medium of different incubations. This correction also takes into account the theoretical maximum abundance of each [^18O]phosphoryl species (i.e.^18O(1), ^18O(2), ^18O(3)) so that the maximum value for the ordinate is 100%. Each value represents the mean of triplicate samples that varied by less than 7%. This is a representative experiment of three that yielded virtually identical results.



Adenylate kinase-catalyzed phosphoryl transfer proceeds at an average rate of 2.1 ± 0.7 nmol bullet mg protein bullet min (n = 4) in non-contracting rat diaphragm muscle (Fig. 1A). In muscle electrically stimulated to contract, AdK catalysis was previously found (1) to accelerate with increasing contractile frequency. Shown in Fig. 1A are the results from a representative experiment in which rat diaphragms were incubated for 4 min in [^18O]water-enriched medium without stimulation to allow a relatively large proportion (80%) of the -ATP to undergo labeling with ^18O while minimal labeling of beta-ADP and beta-ATP occurred. Muscles were then incubated for an additional 2 or 4 min without or with stimulation (1, 2, or 4 Hz). Shown is the rate of AdK-catalyzed phosphorylation of AMP occurring during the second 4-min incubation period. The AdK-catalyzed phosphoryl transfer rate increased 12-fold from 2.1 ± 0.7 to 24 ± 1.2 nmol bullet mg protein bullet min, with stimulation at 4 Hz (the rate per 4 min is shown). At 4 Hz this phosphotransfer rate accounts for about 15% of the total ATP metabolized in terms of the ADP processed by way of AdK catalysis.


Figure 1: The relationship of AdK catalytic rate and the rate of lactate production to frequency of contraction in rat diaphragm muscle. Intact rat diaphragms were isolated and incubated in medium enriched 30-35% in [^18O]water without stimulation for 4 min(1) . During a subsequent incubation in the same medium, the diaphragms were subjected to no stimulation (0 Hz) or electrically stimulated at 1, 2, or 4 Hz. At precisely 4 min after the start of the second incubation period, the diaphragms were rapidly frozen by immersion in liquid nitrogen, and the tissue content of AMP, ADP, ATP, and lactate was determined as described under ``Experimental Procedures.'' The amount of lactate released into the incubation medium was also determined. The rate of AdK-catalyzed phosphoryl transfer was determined for the duration of the second 4-min incubation by the rate of AMP phosphorylation as described previously (1) from the rate of appearance of ^18O-labeled phosphoryls of beta-ATP and beta-ADP. Each value represents the mean of three to four animals analyzed in duplicate or triplicate. The standard errors ranged from less than 1-8% and are presented in the text under ``Results and Discussion'' because most are smaller than the symbols. This experiment is representative of three from which very similar results were obtained. Panel A shows the rate per 4 min of AMP phosphorylation by AdK (designated AMP flux) with and without electrically stimulated contraction at the three frequencies tested compared to the tissue concentrations of AMP, ADP, and ATP. The control value represents the tissue adenine nucleotide level after the 4-min preincubation. The 0 Hz value represents the concentration after the additional 4-min incubation without stimulation. In panel B, rates of AdK catalysis are compared with the rate of lactate production at the contractile frequencies (Hz) shown in parentheses.



These increases in the rate of AdK-catalyzed phosphoryl transfer manifest as rapid phosphorylation of AMP occur with little measurable change in the concentration of muscle ATP, ADP, or AMP (Fig. 1A). In the experiment shown, the cellular concentration of ATP was determined to be (mean ± S.E., n = 3-4) 36.9 ± 0.7, 37.9 ± 1.6, and 35.4 ± 2.4 nmol bullet mg protein after a 4-min period of stimulation at 1, 2, or 4 Hz, respectively, compared to a value of 38.8 ± 1.0 nmol bullet mg protein following a 4-min preincubation of the diaphragm at 0 Hz. The maximum change is an 8.7% decrease in ATP concentration with stimulation at 4 Hz. Similarly, the maximal change in the concentration of cellular ADP was a 35% increase (from 7.36 ± 0.14 to 9.92 ± 0.20 nmol bullet mg protein) after 4 min of stimulating the muscle at 4 Hz while AMP concentration increased 27% (from 1.1 ± 0.04 to 1.4 ± 0.1 nmol bullet mg protein). The ability to detect small changes in the levels of the adenine nucleotides over even a protracted period of vigorous contraction does not eliminate the possibility that larger changes occurred during each contractile event. These results, which are consistent with other reports (9, 10) showing little or no alteration in these nucleotide levels with muscle contraction, nevertheless, indicate that any change that may have occurred in ATP, ADP, or AMP concentration must have been very transient and/or very small and restoration to the basal level very quickly followed each contractile event.

A unique feature of the results shown in Fig. 1A is that although there is no relationship of stimulated muscle contraction with cellular concentrations of adenine nucleotides, there is a close correspondence between contractile frequency and the rate of AdK-catalyzed phosphoryl transfer which is manifest as increases in the rate of AMP metabolic turnover. From the relative constancy of the cellular AMP concentration, it is evident that the enhanced rate of AMP phosphorylation is very closely balanced by and probably coupled to an equivalent rate of AMP generation.

Identification of the Source of the Rapidly Metabolized AMP

How the constancy of this steady state is maintained in the face of such large excursions in flux may be better understood if the catalytic reaction was identified that generates the AMP undergoing metabolic flux in close correspondence with the frequency of stimulated muscle contraction. Identification of the metabolic source of this AMP was at least partially accomplished in earlier experimentation (1) aimed at determining the rate of cellular AMP generation specifically by AdK-catalyzed phosphotransfer compared to other cellular reactions in which AMP is also a product. The latter include pyrophosphorolytic cleavage of ATP in the activation of amino acids and fatty acids, cAMP hydrolytic cleavage by cyclic nucleotide phosphodiesterase, and phosphorylation of adenosine by adenosine kinase (i.e. with [-^18O]ATP). In a muscle equilibrated with [^18O]water, all non-AdK AMP-generating reactions would result in the appearance of AMP, ADP, and ATP with an alpha-phosphoryl oxygen replaced with ^18O. When ^18O appearance in the phosphoryl of AMP and the alpha-phosphoryls of ADP and ATP (i.e. to which the AMP is phosphorylated) was quantitated it was found to proceed at a fixed, relatively slow rate of 0.35 ± 0.03 nmol bullet mg protein bullet min (n = 11). Most importantly, this rate was no different in non-contracting diaphragms than in those stimulated to contract at 1, 2, or 4 Hz when the rate of AMP metabolic turnover represented by the rate of appearance of ^18O-containing beta-phosphoryls in ADP and ATP increased about 12-fold (Fig. 1A). These results clearly define the source of the AMP generated at increasingly greater rates with contractile frequency and undergoing phosphorylation at commensurately increased rates to be ADP transformed to AMP by the catalytic action of AdK.

The next step in defining how this system operates to produce the observed increases in AMP metabolic flux is to identify the source of the ADP from which AMP is generated by AdK-catalyzed phosphotransfer. Identifying this catalytic process can be aided by exploiting a unique feature of the ^18O-phosphoryl-labeling procedure which is that it detects only net flux, represented by newly phosphorylated AMP accumulating as [beta-^18O]ADP and [beta-^18O]ATP. This analytical procedure cannot detect the total number of AdK-catalyzed phosphotransfer events if multiple reversals were to occur because this would not result in a net increase in beta-^18O-labeled nucleotide. The major reactions within eukaryotic cells generating ADP as a product are ATPases and ATP-dependent kinases. Since the rate of this AMP generation closely corresponds to the frequency of stimulated muscle contraction it is most likely that the ADP derives from contraction-related ATPase catalyzed hydrolysis of ATP. This could be designated as the ``AMP flux generating'' reaction.

An additional consideration stemming from the analytical characteristic of the ^18O-phosphoryl-labeling procedure to detect only the net synthesis of newly generated [beta-^18O]ADP and [beta-^18O]ATP is that ATP serving as the phosphoryl donor also cannot derive from an AdK reversal reaction. It must also arise from a process resulting in net production of ATP such as glycolysis or oxidative phosphorylation.

The Metabolic Source of ATP for AMP Phosphorylation

Whether the ATP that provides for the rephosphorylation of this rapidly metabolized AMP derives specifically from one or the other of the two cellular ATP-generating processes was the information sought next. This was first examined by comparing the rate of AdK-catalyzed phosphorylation of AMP with the glycolytic rate in muscle stimulated to contract at increasingly greater frequencies from 0 to 4 Hz. Anaerobic glycolytic rate was assessed by the rate of lactate appearance in the tissue plus that accumulating in the medium. Lactate generation increased in direct proportion to the frequency of contraction (Fig. 1B). The striking relation uncovered is that lactate production proceeds at virtually the equivalent velocity of AdK-catalyzed phosphorylation of AMP. The generation of each of these metabolic products appears to proceed at a fixed rate/contractile event; the total concentration of metabolite formed per unit time is, therefore, proportional to the number (i.e. frequency) of contractile events.

This stoichiometric equivalence between glycolytic flux and rate of AMP phosphorylation (and AMP generation) occurs over the entire range of increased catalytic rates exhibited by each of these metabolic pathways with increasing frequency of contraction. Since lactate production is a measure of anaerobic glycolytic rate, these results indicate a fundamental metabolic relationship between ATP produced by anaerobic glycolysis and AdK-catalyzed transfer of a -phosphoryl of ATP to AMP. Further, the net production of this AMP was traced in the preceding section to ADP generated by a muscle ATPase that had undergone a loss of its beta-phosphoryl as a result of AdK-catalyzed phosphotransfer. Combined, this information demonstrates a functional link by way of AdK-catalyzed phosphotransfer between ATP consumption by an ATPase and ATP generation by anaerobic glycolysis. There is abundant evidence that the paradoxical generation of lactate in fully oxygenated tissues (11) represents a constitutive process for generating ATP utilized selectively by specific cellular components(12, 13) . This view is also consistent with the recent observation using C NMR (14) that two pyruvate pools exist in muscle cells, one of which is associated with nonoxidative metabolism (i.e. lactate formation).

Relationship of Adenylate Kinase-catalyzed Phosphoryl Transfer and Enhanced Glycolytic Flux in Muscles with Impaired Oxidative Metabolism

The basis of the nearly equivalent relationship uncovered in contracting muscle between lactate-producing glycolysis and AdK-catalyzed phosphoryl transfer resulting in rapid net increases in the rate of AMP phosphorylation was further probed by determining the relationship of these metabolic processes when anaerobic glycolytic rate is accelerated as a compensatory means of maintaining cellular high energy phosphate metabolism. To accomplish this, oxidative production of ATP was suppressed by depriving the muscle of oxygen (using nitrogen-equilibrated medium) by treating the muscle with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to uncouple oxidative phosphorylation or by treatment with KCN to inhibit oxidative phosphorylation.

In the oxygen-deprived diaphragms, the rate of ^18O-labeled phosphoryl appearance in beta-ADP and beta-ATP was greatly increased compared to fully oxygenated controls (compare Fig. 2, A and B). This increase occurred with virtually no change in the rate of [^18O]phosphoryl appearance in -ATP. This indicates that the overall rate of ATP metabolism with respect to its energy-rich -phosphoryl was not impaired. These results also demonstrate that the large increase in the rate of [^18O]phosphoryl appearance in beta-ADP and beta-ATP is the outcome of an increase in the rate of AdK catalysis rather than a change in ^18O enrichment of the -ATP phosphoryl donor.

Suppression of oxidative phosphorylation by each of these experimental procedures produced the predicted effect of enhancing anaerobic glycolytic flux; the rate of lactate generation was increased in the range of 8-26-fold (Fig. 3). Another outcome of impaired oxidative metabolism was an enhancement in the rate of net AdK-catalyzed phosphorylation of AMP which increased by approximately the same magnitude as the rate of anaerobic glycolysis (Fig. 3). The basal rate of AdK-catalyzed phosphoryl transfer resulting in AMP phosphorylation increased approximately 20-, 25-, and 8-fold with N(2), FCCP and KCN, respectively. These net increases in AdK-catalyzed phosphotransfer coincided with nearly equivalent increases of about 16-, 26-, and 8-fold in anaerobic glycolytic flux for N(2), FCCP, and KCN, respectively (Fig. 3).


Figure 3: The relationship of suppressed oxidative phosphorylation to the rates of AdK catalysis and lactate production. Non-stimulated rat diaphragms were incubated with no addition to the medium or with addition of an agent that suppresses mitochondrial oxidative phosphorylation. Tissues receiving KCN (0.5 mM) and FCCP (10 µM) were exposed to the inhibitors for 5 min prior to their transfer to [^18O]water-containing medium. The tissues incubated in N(2)-equilibrated medium were treated as described in Fig. 2. The AdK velocities and lactate production rates were calculated from determinations at 1, 2, and 4 min of incubations in the [^18O]water-enriched medium. The values shown represent the mean rates ± S.E. determined from at least four diaphragms. Lactate present in tissue incubated for only the 5-min preincubation period was subtracted from the total lactate in tissues subjected to the subsequent incubation. Lactate present in the [^18O]water-containing medium was included in the total. Tissue adenine nucleotide concentrations were also determined in diaphragms incubated for 2 and 4 min in [^18O]water-containing medium, and the mean values ± S.E. are presented in the text.



In each of these experimentally induced states of suppressed oxidative phosphorylation, the increases in anaerobic glycolytic generation of ATP must have compensated reasonably well for the diminished rate of ATP production oxidatively because the total ATP utilization rate determined from ^18O incorporation into metabolic intermediates (2) was reduced (19%) only after treatment with KCN (not shown). In contrast to the near equivalent relationship uncovered between AMP metabolic flux and anaerobic glycolytic rate (Fig. 3), changes that occurred in the cellular concentrations of adenine nucleotides under these different conditions of impaired oxidative ATP production showed little correspondence with the magnitude of enhanced glycolysis. The correspondence sought in this instance was a change in a metabolite effector (i.e. ATP, ADP, and/or AMP) concentration that would elicit an 8-26-fold acceleration in the velocity of a rate-controlling glycolytic enzyme. For example, according to predictions of allosteric regulation (9, 15) a change of 400% in metabolite effector concentration is required to produce a 9-fold increase (i.e. the smallest increase observed in gycolytic flux) in enzyme velocity. The total cellular concentration of ATP was decreased from a control value of 46.4 ± 4.6 nmol bullet mg protein (n = 4) by 5.4, 20.4, and 14.5% with N(2), FCCP, or KCN treatment, respectively. The concentration of ADP (control value 12.7 ± 3.0 nmol bullet mg protein, n = 4) increased by 26, 71, and 31.6% with N(2), FCCP, or KCN treatment, respectively. The cellular concentration of AMP (control value 2.2 ± 0.9 nmol bullet mg protein, n = 4) increased by 27.5, 126, and 92% with N(2), FCCP, or KCN, respectively. Changes of similar magnitude in adenine nucleotide concentrations have been observed with comparable treatments that result in suppression of muscle oxidative ATP generation(16) . If or how these altered levels in adenine nucleotides may contribute to the observed accelerated glycolytic flux is not readily apparent from the magnitude of their changes relative to those of glycolytic flux. For example, the decrease in the concentration of ATP and increases in ADP and AMP concentrations were greater with KCN treatment than with N(2) replacement, but the increase in gycolytic flux was more than two times greater with N(2) than KCN. The possibility that localized changes in nucleotide concentration represents a determinant of the observed increases in glycolytic flux has not been eliminated by these results.

On the other hand, the action of each treatment that suppressed oxidative ATP generation resulted in an accelerated rate of AMP metabolic flux that exhibited a very high fidelity with anaerobic glycolytic flux. This correspondence was manifest as a stoichiometry whereby each molecule of ATP generated as a result of the catabolism of triose phosphate to lactate coincides with an almost equivalent phosphorylation of an AMP molecule. This striking fidelity of AdK-catalyzed AMP phosphorylation to anaerobic glycolysis is, therefore, demonstrable in the nearly physiological state with intermittent stimulation of muscle contraction (Fig. 1, A and B) and also under experimental conditions whereby oxidative production of ATP is impaired continuously (Fig. 3).

The Relationship between AdK and Creatine Kinase Catalysis in the Intact Muscle Cell

Substantial evidence has accumulated in support of a role for creatine kinase-catalyzed phosphoryl transfer providing a cellular high energy phosphoryl shuttle capability (17, 18, 19) . If this represents the metabolic importance of creatine kinase, then its behavior in relation to AdK-catalyzed phosphotransfer, for which a similar functional importance has been proposed(1, 4, 5) , warrants examination especially when the activity of the AdK system is accelerated as, for example, in the state of impaired oxidative production of ATP during oxygen deprivation.

The metabolic behavior of creatine kinase in intact muscle is assessable from the rate of ^18O appearance in the phosphoryl of CrP. This results when the phosphoryl of [-^18O]ATP is transferred to creatine to generate [^18O]CrP. When examined by this analytical approach it was very evident that the kinetic behavior of creatine kinase in intact muscle deprived of oxygen exhibited reciprocal behavior relative to that of AdK. For example, when the muscle was deprived of oxygen and AdK-catalyzed phosphotransfer increased about 20-fold (Fig. 3), there was a decrease in the rate of appearance of [^18O]CrP by about 65% (Fig. 4A). Contributing to this diminished velocity of CK catalysis was an approximately 60% reduction in the concentration of cellular CrP when oxidative generation of ATP was impaired by N(2) equilibration of the incubation medium (Fig. 4, B and C). This reduction in the level of CrP occurred during the 4-min preincubation in nitrogen-equilibrated medium and reflects a new steady state that was sustained during the subsequent incubations of 1, 2, or 8 min in nitrogen-equilibrated [^18O]water-enriched medium. As shown in Fig. 4C, the reduced concentration of CrP in the oxygen-deprived muscle undergoes metabolic turnover (i.e. acquires ^18O-labeled phosphoryls) at an apparently pseudo-first-order rate nearly identical to that of the approximately two times greater CrP concentration in the oxygenated tissue (t = 3.83 and 3.90 min in oxygenated and N(2)-equilibrated tissue, respectively). The metabolic flux of CrP, which is equal to the turnover rate times its cellular concentration, is reduced by approximately the same magnitude as the cellular concentration of CrP.


Figure 4: Effect of oxygen deprivation on creatine kinase-catalyzed generation of [^18O]creatine phosphate and the cellular concentration and metabolic turnover of creatine phosphate. Creatine phosphate was purified as described previously (7) from the same tissues used to obtain the data shown in Fig. 2, and the ^18O content of its phosphoryl was determined after its enzymic transfer to glycerol (i.e. glycerol 3-phosphate) using CK and glycerol kinase. The values shown are means from three diaphragms. Values from replicate tissues varied by less than 10%. Panel A shows the relative rate of CK-catalyzed phosphoryl transfer (i.e. appearance of [^18O]creatine phosphate) in fully oxygenated tissues (circle) and in tissues incubated in medium depleted of oxygen (bullet). The ordinate represents the product of the tissue concentration of creatine phosphate times the fraction of the total cellular creatine phosphate with a phosphoryl containing one to three atoms of ^18O (corrected for the enrichment of [^18O]water). Panels B and C show the tissue concentrations of creatine phosphate (striped portion) and the fraction containing an ^18O-labeled phosphoryl (filled portion) in diaphragms incubated in fully oxygenated medium (panel B) or nitrogen-equilibrated medium (panel C).



This decrease in CrP concentration is most likely a consequence of the diminished rate of ATP generation by oxidative phosphorylation apparently linked to CK-catalyzed phosphorylation of creatine. Since the cellular ATP concentration is sustained at very nearly the control level (i.e. 46.4 ± 4.6 versus 43.9 ± 2.8 nmol bullet mg of protein, n = 4), and the ATP metabolic flux is not compromised in the oxygen-deprived muscle (Fig. 2, A and B), both are probably maintained in large measure by the compensatory increase observed in anaerobic glycolytic ATP generation. This increased rate of anaerobic ATP generation indicated by the enhanced rate of lactate production occurs as was pointed out above (Fig. 3) in conjunction with an almost equivalent increase in the net rate of AdK-catalyzed phosphotransfer. This increase in AdK-catalyzed phosphotransfer could, therefore, be viewed as compensatory phosphotransfer for the diminished rate of CK-catalyzed phosphoryl transfer coinciding with the reduced rate of oxidatively generated ATP in the oxygen-deprived muscle.

The concept that CrP is simply a reservoir of energy-rich phosphoryls used to maintain or ``buffer'' the cellular ATP concentration when ATP production does not keep pace with its utilization could not explain the results reported here. A constantly maintained cellular concentration of CrP exhibits a relatively rapid rate of turnover under basal conditions and continues to exhibit virtually the same turnover even in the oxygen-deprived state when the cellular CrP concentration is markedly compromised, but maintained at a relatively constant diminished steady state level.

These results support the conclusion that in skeletal muscle AdK-catalyzed phosphotransfer is linked to anaerobic glycolytically produced ATP and that catalyzed by CK involves oxidatively produced ATP. These results also indicate that the AdK and CK phosphotransfer systems share a similar metabolic function, and although they can operate independently of one another their functions are interrelated to the extent that AdK may replace the phosphotransfer function of CK.

Justification for Assigning Sequential AdK-catalyzed Reactions the Function of High Energy Phosphoryl Transfer and Metabolic Regulation

The suggested mechanism by which the AdK-catalyzed phosphoryl transfer system may function in skeletal muscle is shown in Fig. 5. This model is based in part on the Mitchell (20) principle of vectorial ligand conduction. The concept of vectorial ligand conduction (21) was originally put forth to provide a mechanism for transporting ligands within the domains of enzyme catalytic centers and across lipid bilayers. In the modified version presented here, it is envisaged to provide a mechanism for bidirectional conduction of energy-rich - and beta-phosphoryls through a soluble, near equilibrium system in one direction and an AMP signal for promoting glycolytic ATP production in the other direction. According to this concept, displacement of an equilibrium at one end of a series of enzyme-catalyzed equilibrium reactions results in rapid bidirectional relaying of the conducted species (i.e. energy-rich phosphoryl or spent metabolite) to a distal site. This metabolic wave propagation has been calculated to proceed at a rate that can be orders of magnitude faster than the diffusion rate of reactants(22, 23) . The ligand conduction mechanism also contrasts with the traditional diffusion theory by operating independently of cytosolic ligand concentrations and without movement of a specific ligand through the entire length of the pathway(24, 25) .


Figure 5: Suggested functional arrangement of the proposed adenylate kinase and the creatine kinase-catalyzed high energy phosphoryl transfer systems. The scheme shows a proposed mechanism of communication between flux-generating (ATPases) and flux-responding (glycolysis or oxidative phosphorylation) processes by way of sequential transfer of phosphoryls catalyzed by a linear sequence of adenylate kinase (AdK) (designated A) or creatine kinase (CK) (designated B) enzyme molecules comprising an energy transfer and regulation network in the cellular cytosol. The proposed model, which incorporates the previously suggested ``PCr circuit'' construct(19) , emphasizes relaying the displacement of equilibrium along a corresponding chain of enzyme molecules according to the Mitchell (20, 21) principle of vectorial ligand conduction. Incoming ligands ``push'' others to create a ``pressure'' that is propagated through the network of enzymes catalyzing rapid equilibration among ligands; the molecules arriving at the distal sites of this bidirectional system represent the equivalent rather than the specific molecule generated at the originating site. A mechanism similar in principle has been proposed for proton conduction pathways (e.g. ``proton wires'') across biological membranes(25) . The reaction sequence designated A shows how AdK could function at the site of an ATPase to restore a molecule of ATP and simultaneously generate a signal in the form of a molecule of AMP to promote glycolytic production of a new molecule of ATP to replace the molecule of ATP consumed. This AMP signal can enter the AdK-catalyzed transfer system and push monophosphate molecules distally along a series of rapidly equilibrating enzymes responding to the disequilibrium created at the ATPase site. This AMP promotes the emergence of an equivalent at the end of the transfer system distal to the flux generating ATPase and in close proximity with an as yet unidentified glycolytic site. It can promote glycolytic production of an ATP molecule by serving as phosphoryl acceptor of a -phosphoryl from an ATP molecule acting as an inhibitor of this control site in glycolysis. The reaction sequence designated B shows how the previously proposed PCr circuit (19) would be integrated according to the experimental results described here with the operation of the AdK ligand conduction system. The high energy phosphoryl represented by the -phosphoryl of an ATP molecule generated by oxidative phosphorylation is ordinarily conducted through a series of phosphotransfers in the form of CrP or ATP. However, if oxidative phosphorylation becomes impaired, viz.B(1), the ADP from the ATPase ordinarily rephosphorylated by CrP through CK catalysis becomes a reactant in the AdK-catalyzed phosphotransfer system. This results in the generation of an AMP signal that promotes anaerobic glycolytic flux as a compensatory means of generating ATP along with a commensurably greater rate of AdK-catalyzed transfer of this ATP.



The evidence in support of this proposed scheme is as follows. The initiating or ``flux generating'' reaction is designated as a localized cellular ATPase. This was concluded because the AMP-generated and undergoing phosphorylation was traced to an ADP reactant in an AdK-catalyzed phosphotransfer reaction. Furthermore, this ADP was determined to derive from a cellular reaction generating net ADP in close correspondence with a contraction-related process very likely represented by an ATPase activity. ATP generation from anaerobic glycolysis is designated as the source of net ATP used for AMP phosphorylation because this process exhibited equivalent stoichiometry with that of AMP phosphorylation. Additionally, inhibition of oxidative phosphorylation resulted in markedly enhanced rates of anaerobic glycolysis coinciding, quantitatively, with AdK-catalyzed phosphotransfer. Consistent with this conclusion is that AdK and glycolytic enzymes are colocalized in muscle fibers(26, 27, 28) . One of the two molecules of ADP generated at this glycolytic site by AdK-catalyzed phosphotransfer is envisaged to represent a substrate and/or allosteric effector that can promote glycolytic flux and generation of another ATP molecule. The other molecule of ADP is considered to represent a high energy component due to its energy-rich beta-phosphoryl which is transferable exclusively by the catalytic action of AdK along the equilibrating sequence of AdK-catalyzed reactions as the beta- or -phosphoryl of ADP or ATP to the site of the ATPase where it can regenerate a consumable molecule of ATP.

The suggestion that this anaerobic glycolytically produced ATP may serve as a suppressor of glycolysis is based on the logic that ATP should regulate its own synthesis. In addition, there are at least three identifiable sites in the glycolytic pathway, phosphofructokinase, aldolase, and glyceraldehyde phosphate dehydrogenase, that are inhibitable (i.e. allosterically) by ATP(29, 30, 31) . There is evidence that AdK can interact with phosphofructokinase (32) and also with aldolase, phosphoglycerate kinase, and enolase which copurify with AdK on a Sepharose-Ap5A column. (^2)Recently, it was found that in Escherichia coli AdK is associated with glyceraldehyde phosphate dehydrogenase. (^3)

Under consideration are two possible mechanisms by which this putative glycolytic inhibitory action of ATP could be reversed. First, the ATP acting as glycolytic suppressor could be bound to an ATP-inhibitable site and in rapid equilibrium with a localized pool of ATP. The concentration of ATP in this locale would become decreased by the AdK-catalyzed conversion of this ATP to ADP after arrival of an equivalent AMP.

A second possibility is that AdK may itself act as an effector protein by analogy with G-proteins (e.g. ras p21) with which it shares structural homology(33, 34) . AdK, like G-proteins undergoes very marked conformational changes upon binding their triphosphate ligand(34, 35) . In the case of adenylate kinase, there is sufficient cytosolic ATP to maintain AdK in the triphosphate-liganded state which may represent a conformation imparting a suppressive influence on the glycolytic enzyme activity in question. Upon delivery of AMP and conversion of ATP to ADP, resulting from catalytic phosphotransfer, transformation from an inhibitory to a non-inhibitory conformational state would occur. The magnitude of deinhibition would, therefore, depend on the frequency of AMP generation. This mode of regulation would be much more rapid than can be accomplished with G-proteins which exhibit ``on/off'' time constants of several seconds due to their inherently slow phosphohydrolase activity(36) . This would compare to less than a millisecond time constant required for the adenylate kinase catalytic cycle. A precedent for this type of action for AdK can be inferred from results (37) showing that a mutant form of AdK imparts thermolability to another enzyme (e.g.sn-glycerol-3-phosphate acyltransferase) while the ``normal'' AdK is required for a stimulatory effect of ATP on this enzyme activity. A recently uncovered AdK-catalyzed AMP metabolic dynamic similar to that described here but with high fidelity to the opening and closing of an ATP-inhibitable K channel in insulin secreting transformed pancreatic beta-cells (38) would also be consistent with a regulatory role for the AdK enzyme protein in the function of K channel regulated by apparent ATP/ADP liganding(39) .

The possibility of a series of AdK-catalyzed equilibrium reactions is suggested by comparing the present results with a report of the velocity of AdK-catalyzed phosphotransfer measured by P NMR saturation transfer(40) . The rate determined by the latter procedure is approximately 20-fold greater than that determined by the procedure of [^18O]phosphoryl labeling in muscle and in red blood cells(1, 41) . The P NMR method theoretically detects virtually all AdK-catalyzed phosphotransfers occurring during the monitoring period, while the latter only detects net AMP phosphorylation by newly synthesized ATP. The simple interpretation of these apparently conflicting results is that each newly generated ADP by AdK catalysis undergoes approximately 20 sequential phosphotransfers in a ligand conduction-type of system.

That oxidatively generated ATP is transferred primarily by the CK-catalyzed phosphotransfer system is strongly suggested by the decreased rate of [^18O]CrP generation accompanying suppression of oxidative ATP production. This decreased oxidative generation of ATP paralleled by diminished CK-catalyzed phosphotransfer occurred in conjunction with a markedly enhanced rate of AdK-catalyzed phosphotransfer and a correspondingly increased rate of anaerobic glycolytic generation of ATP. Relevant to this issue is a subsequent observation, which will be the subject of a separate report, that the net rate of CK-catalyzed phosphotransfer in non-contracting rat diaphragm muscle is equal to about 85% of the total ATP metabolic turnover. Under the same conditions AdK-catalyzed phosphotransfer accounts for less than 10%. However, when CK activity is progressively suppressed in the intact muscle by increasing dinitrofluorobenzene concentrations the activity of the AdK phosphotransfer system progressively increases to the extent that when CK activity is 98% inhibited, AdK accounts for the transfer of virtually 100% of the ATP produced(42) . Under these circumstances over 80% of the ATP continues to be generated by oxidative metabolism. This indicates that AdK can also transfer oxidatively produced energy-rich phosphoryls when CK-phosphotransfer system is directly impaired. The previously reported coexistence of AdK and CK in discrete subcellular locales (26, 43) is consistent with this shift from one phosphoryl transfer system to the other.

The CK- and AdK-catalyzed phosphotransfer systems therefore appear to share a similar metabolic function with respect to energy-rich phosphoryl transfer. Under certain physiological conditions such as at high muscle workloads when CrP concentration undergoes significant reduction (44, 45) AdK could be predicted to take over a substantial portion of this function when it cannot be carried out by a markedly constrained CK phosphotransfer system.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant GM28818 and the Minnesota Leukemia Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 612-625-1118; Fax: 612-625-2163.

(^2)
P. P. Dzeja, R. J. Zeleznikar, M. M. Moos, Jr., and N. D. Goldberg, unpublished observation.

(^3)
M. Glaser, personal communication.

(^1)
The abbreviations used are: AdK, adenylate kinase; CK, creatine kinase; CrP, creatine phosphate; ^18O, oxygen-18 isotope; FCCP, p-(trifluoromethoxy)-phenylhydrazone.


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