Role of the Adenylate Deaminase Reaction in Regulation of Adenine Nucleotide Metabolism in Ehrlich Ascites Tumor Cells1

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[CANCER RESEARCH 36, 1144-1 150, March 1976] Role of the Adenylate Deaminase Reaction in Regulation of Adenine Nucleotide Metabolism in Ehrlich Ascites Tumor Cells1

Astrid G. Chapman, Arnold L. Miller,2 and Daniel E. Atkinson

Biochemistry Division, Department of Chemistry, University of California, Los Angeles, California 90024

SUMMARY ing cell by regulating the rates of ATP-utilizing and ATP regenerating sequences have been discussed (1-3) and are The regulatory properties of adenylate deaminase (EC implicit in the subsequent discussion. 3.5.4.6) from Ehnlich ascites tumor cells suggest that the It has been known for a number of years that upon the reaction catalyzed by this enzyme serves to protect the cell addition of glucose on 2-deoxyglucose to incubated ascites against sharp decreases in the adenylate energy charge by cells there is a rapid and eventually reversible decrease in removing adenosine 5'-monophosphate generated when both the concentration of ATP and the total adenine nucleo the rate of utilization of adenosine tniphosphate is suddenly tide concentration of the cells (13, 15, 16, 21, 22, 25, 35). In increased. The enzyme is effectively inhibited under normal Chart 1, the analytical results of Overgaard-Hansen (25) are physiological conditions of high energy change (0.9) and 4 plotted, together with the calculated values of the adenylate to 5 mM adenine nucleotide pool size. The reaction is energy charge. Conversion of AMP to inosine has recently sharply activated by a decrease in the energy change in the been shown to occur mainly via the AMP deaminase neac physiological range (0.9 to 0.6). At low energy change (0.6), tion, followed by a dephosphorylation of IMP (22). decrease in the size of the pool causes a marked and non The utilization of ATP and the subsequent transient in linear decrease in the rate of the deaminase reaction. This crease in AMP can, of course, be explained in terms of the effect presumably serves to prevent excessive depletion of sudden high rate of hexokinase-mediated phosphorylation the adenine nucleotide pool. Calculations based on the of the added carbohydrate, followed by a rapid conversion kinetic data obtained in this study show that the AMP deam of the ADP produced to AMP and ATP through the action of inase reaction can account for the well-established altena adenylate kinase, but it has remained unclean why there tion of adenine nucleotide metabolism that is observed should be a concomitant decrease in the level of the total following addition of glucose on 2-deoxyglucose to intact adenylate pool. This question is compounded by the fact ascites cells. that AMP deaminase from ascites cells, as from other sources, has been shown to be activated by ATP (4, 35), but the increase in the rate of the reaction following glucose INTRODUCTION addition to the cells coincides with a decrease in the level of the activator ATP. Adenine nucleotide metabolism in Ehnlich ascites cells It has been suggested that the depletion of the adenylate has been studied extensively, partly in search of a clue to pool is caused by a release of the known phosphate inhibi the high rate of aerobic glycolysis in these tumor cells. The tion of AMP deaminase as the intracellular phosphate level total concentration of the adenine nucleotides rn ascites decreases (35). This undoubtedly contributes to the negula cells is normally maintained at a steady level (in the range tion of the enzyme, but the drop in the adenylate pool is 3.5 to 5 p@moles/mlof cells) under different conditions of observed even when the concentration of phosphate in the growth and incubation by keeping the rates of synthesis and medium is very high (25) and a smaller decrease in the utilization of the adenine nucleotides in balance (12, 17, 18, intracellular phosphate concentration would be expected. 20-22, 24, 25, 33, 35). Within the adenylate pool there is an We have recently suggested that a very similar fructose extremely rapid interconversion of the 3 adenine nucleo induced adenylate depletion in liver serves to stabilize the tides, yet the adenylate energy charge (ATP + 0.5 ADP)/ adenylate energy charge (8). When the energy demand on (ATP + ADP + AMP) (1) is maintained at a value of about the cell is suddenly increased, the AMP that would tend to 0.90 in ascites cells (12, 17, 18, 20-22, 24, 25, 27, 33, 35) as accumulate is rapidly removed by the AMP deaminase neac well as in most tissues and organisms studied (9). The need tion, thereby restoring the energy charge to control values. for a closely controlled energy charge value and the role of This response is then followed by a much slower replenish thisparameterinmaintaininghomeostasisina metaboliz ing of the adenine nucleotide pool. I This work was supported in part by Research Grant AM09863 from the The energy change values shown in Chart 1 suggest that National Institute of Arthritis, Metabolism, and Digestive Diseases. this proposal can be extended to ascites tumor cells. This 2 Recipient of Postdoctoral Fellowship 5 FO2-CA-28,649 from the National paper reports experiments on AMP deaminase from ascites Cancer Institute. Present address: Department of Neurosciences, University of California, San Diego, La Jolla, Calif. 92037. cells. The results are consistent with the prediction that the Received December 13, 1974; accepted December 3. 1975. regulatory properties of this enzyme contribute to the ob

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was dissolved in Tnis-glycerol buffer (3 ml) and stored at 5 i0 â€” 1 5Â°. The enzyme was purified only about 1 2- to 1 5-fold 4 La 2 with a 40 to 50% yield, but the preparation was free of 0 adenylate kinase and ATPase activities. In this form, the 2e c@ItÂ°@;@ 4 n I enzyme was stable for 10 to 15 days at â€”15Â°.However the U.! â€˜4 0 response of the enzyme to ATP activation decreased toward z@ 7, \ .2 the end of this period, and consequently the kinetic studies were carried out within 1 week of the enzyme purification. 0 2 4 6'1E 3@ 50 TIME (minI Freezing, which was prevented at â€”15Â°bythe presence of Chart 1. Changes in concentrations of ATP, total adenine nucleotides (@ glycerol, caused a 20 to 50% inactivation of the enzyme Ad.N.), inosine, and adenylate energy charge (E.C.) values in Ehrllch ascites activity. tumor cells following addition of 5 m@ glucose at zero time. Tumor cell Assays for AMP Deamlnase Activity. The glutamate de volume was 7.8% and the cells were suspended in 55 m@@iphosphate-Locke's solution (pH 7.35) at 37Â°.From the data of Overgaard-Hansen (25). hydnogenase-coupled determination of the ammonium gen erated (8) was routinely used in the kinetic studies. A stand served glucose-induced variations in the adenylate concen and assay mixture for measuring optimal enzyme activity trations in the intact cell. contains: 100 mM imidazole chloride, pH 6.8, 100 mM KCI, 4 mM AMP, 2 mM ATP, 4 mM MgSO4, 4 mM a-ketoglutarate, 0.26 mM DPNH, and 0.5 mg of commercial glutamate dehy MATERIALS AND METHODS drogenase (Boehninger enzyme in 50% glycerol). The assay mixture, including glutamate dehydrogenase, Ascites Cells. Ehrlich ascites cells were grown in 10- to was preincubated at 37Â°for10 to 15 mm before the reaction 12-week-old Swiss female mice and collected on the 8th day was initiated by the addition of 10 to 50 @gof partially following a 0.25-mI i.p. inoculation. The average yield was purified AMP deaminase. The reaction was followed at 340 about 4 to 6 ml of packed cells pen animal. After the cells nm by means of a Vanian Model 135 spectrophotometen with were collected in a beaker containing hepanin, they were recorder. washed twice with 0.9% NaCI solution to remove erythro During the purification, the AMP deaminase activity was cytes. Following a subsequent wash in 50 mM Tnis-CI, pH measured by the microdiffusion method (28). The amount of 7.4, 5 mM /3-mercaptoethanol, and 20% glycerol (Tnis-glyc ammonia generated in 30 mm at 37Â°wasdetermined colon erol buffer), the cells were suspended in the initial volume of metrically at 420 nm after reaction with Nessler's reagent. @ the same Tris-glycerol buffer. In order to determine a possibleeffect of or DPNH on The cells were broken by sonic disruption (Bronson sonic the AMP deaminase reaction, the conversion of AMP to IMP oscillator at 8 amp) for a total of 2 to 2.5 mm (in 20-sec was followed directly at 285 nm (19). Because of the high periods interspersed with 1-mm cooling periods). The cell extinction coefficient of the adenine nucleotides at 285 nm, debris was removed by 60 mm of centnifugation at 40,000 x the assay can be used only when the total adenylate con g, and the cell extract was stored frozen at â€”20Â°. centration is less than about 4 mM, which corresponds to an Partial Purification of AMP Deaminase. The AMP deami absorbance of 2.8 at 285 nm. nase activity from ascites cells was completely stable for 2 Calculation of Total Mg2@Concentration Corresponding years when the crude cell extract was stored at â€”20Â°.How to I m@Free [email protected] the amount of Mg2@thatis even, subsequent purification led to a fairly rapid inactiva complexed by the different components in the assay mix tion of the enzyme. tune, the following stability constants, quoted by Blair (6), AMP deaminase from ascites cells was purified by a pro were used: MgATP2, 73300 N' ; MgHATP, 600 M'; cedure similar to that used for the liver enzyme (8). The MgADP, 4290 M'; MgHADP, 95 M'; and MgAMP, 90 M@. enzyme activity in the crude cell-free extract (50 ml) was Veloso et a!. (32) assumed the stability constant of the Mg - precipitated with 2 M Li 250 4. Following centnifugation at a-ketoglutanate complex to be the same as that reported for 40,000 x g for 20 mm, the Li 250 4 precipitate was suspended the Ca - a-ketoglutarate complex, namely 19.5 M. The in a minimal amount of Tnis-glycerol buffer (10 ml) and stability constant used for the Mg - citrate complex, 1995 dialyzed for 14 hr against 4000 ml of 10 mt@iTnis-CI,pH 7.4, M', is also quoted by Veloso et a!. The KATP@ complex (K,

and 5 mM /3-mercaptoethanol. The volume of the enzyme = 14 M@) and the KADP2 complex (K, = 4.8 M'), as well as increased 2.5-fold during dialysis. The dialyzed enzyme was complexing of Mg2@by other components of the reaction, immediately transferred to a DEAE-cellulose column (diam have been ignored. The values were not corrected for tem eter, 2.5 cm; height, 42 cm) preequilibrated with 50 m@Tnis penatune. Cl, pH 7.4, and 5 mM /3-mencaptoethanol. Approximately 4.5 The pK values used in the calculations were: ATP, 7.05; to 5 mg of protein were used pen ml of column material. The ADP, 6.78; and citrate, 6.4. When these values are used, enzyme was eluted with a linear ionic strength-pH gradient, Equation A reduces, at pH 6.8 and 1 mM free Mg2@in the consisting of 450 ml of 50 mti Tnis-CI, pH 7.4, and 5 mM @3- absence of citrate, to Equation B. mercaptoethanol in the mixing chamber, and 450 ml of 75 mM citrate, pH 6.8, and 5 mM /3-mencaptoethanol in the Mg@1 = Mg@ + Mg - a-ketoglutarate + MgATP2 A eluting chamber. The enzyme eluted at approximately 650 ml of eluent volume, and the peak fractions (90 ml) were + MgHATP + MgADP + MgHADP + MgAMP

@ immediately made 2 M in Li 2SO @.Theprecipitated enzyme (+ Mg citrate, when applicable)

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Mg@,,1= 1.077 + 0.595 (ATP)@OtaI B

+ 0.456 (ADP),,@,1 + 0.083 (AMP)totaj

RESULTS

The properties of AMP deaminase from Ehrlich ascites tumor cells are very similar to those of the liven enzyme. In the absence of the activator ATP, the enzyme has a very low affinity for the substrate AMP. The sigmoidal substrate satu ration curve is shown in Chart 2,4. Increasing the concentra tion of ATP increases the affinity of the enzyme for the substrate without changing the maximal velocity of the re Chart 3. Activation of ascites AMP deaminase by ATP. The concentration of AMP was 1 mM, and the concentration of free Mg'@ was 1 [email protected] action, as seen in Chart 2B. The inset tabulates the S0.5 remaining conditions are described in the legend to Chart 2. values for AMP at different ATP concentrations and shows that the slope of a Hill plot, m, changes from about 2 in the absence of ATP to about 1 in the presence of ATP at con C @ centrations of 1 mM on higher. It can also be seen from Chart (I 2 that the maximal response of the enzyme to variations in substrate concentration occurs at concentrations of AMP @a) normally found within the cell (0.1 to 0.3 @mole/mIofcells) O@ C when ATP is present at physiological concentration. At physiological substrate concentrations, ATP activates the AMP deaminase reaction approximately 100-fold (Chart zj 3). The value of M0@5(themodifier concentration at half maximum activation) and the Hill slope for the ATP activa tion were calculated to be 1.6 mM and 1.8, respectively. We 0 2 4 6 8 10 12 14 observed some variation with respect to the ATP activation IMgSO4] mM between different preparations of the enzyme (range of M0.5 Chart 4. Rate of the reaction catalyzed by ascites AMP deaminase. The @ values observed: 1.2 to 1.7 mM), but the response was reaction mixture contained 1 mM AMP, 2 [email protected],and as indicated. The remaining conditions are described in the legend to Chart 2. always sigmoidal. Atkinson and Murray (4) previously ne ported a greater affinity for ATP of the Ehrlich ascites en approximately equal to the level of the nucleotides present. zyme (M0@5=0.07 mM, and a 10-fold activation by ATP at 0.6 Beyond this point, any further increase in the Mg2@concen mM AMP). This discrepancy might be explained by the ab tnation causes a reversal of the ATP activation. Since similar sence of magnesium in their assay mixture; we have found that Mg2@is required for maximal ATP activation. Higher results were obtained with ,, the anion does not appear to cause the observed inhibition. In the absence of ATP, the concentrations of Mg2@will reverse this activation (Chant 4). In the absence of Mg2@andat a substrate concentration of 1 reaction does not require Mg2@foroptimal activity, and high Mg'@ (20 mM) causes only a 10 to 15% inhibition of the mM, the activity is increased 15- to 20-fold by the addition of 2 mM ATP. Increasing concentrations of Mg'@increase the activity observed with 10 m@iAMP. Because of the sensitivity of the enzyme to variations in degree of ATP activation until the Mg2@concentration is the concentration of Mg'@, we attempted in most of the experiments to maintain the concentration of free Mg2@at1

c250 ABâ€”.--@ mM. This, according to measurements made in intact cells, appears to be the approximate level of uncomplexed mag nesium found in ascites cells and other mammalian cells (16, 32). The concentration was adjusted on the basis of @/(._@T/@:rcalculations of the amount of Mg2@complexed by the differ ent nucleotides and the other components of the assay @ 10 I TP@,@*00534 /â€¢ â€˜ 1 @1':'1@I'@1H@ mixture as described in â€œMaterialsandMethods.â€•Underthe 1/ conditions used in the experiments summarized in Chart 4, z @ / â€˜â€˜â€˜â€˜,1 Pr . a free Mg2@concentration of 1 m@ corresponds to a total 0 10 20 30 40 500 1 2 3 4 5 6 Mg2@concentration of 2.35 mM, which is within the range of AMP mM AMP mM concentrations causing the maximal plateau in the activa Chart 2. Rate of the reaction catalyzed by ascites AMP deaminase as a function of AMP concentration in the absence (A) and presence (B) of ATP at tion curve. different concentrations. The reaction mixture contained 100 m@iimidazole The reversal of the ATP activation by high concentrations chloride buffer, pH 6.8, 100 mM K@,0to 4 mM ATP where specified, AMP as indicated, and 1 mM free Mg'@ calculated as described in â€œMaterialsand of Mg2@is in contrast to the results obtained with AMP Methods.â€•Themixture also contained 4 mM a-ketoglutarate, 0.26 mM DPNH, deaminase from liven. The liven enzyme required Mg2@for and 0.5 mg commercial glutamate dehydrogenase. The reactions were car maximal ATP activation, but excess Mg2@did not affect the ned out in 1 ml volume at 37Â°asdescribed under â€œMaterialsandMethods.â€• The inset shows calculated 5,., values and Hill slopes, m, at different ATP activity further. concentrations. When no ATP is present, ADP is an activator of the AMP

1146 CANCER RESEARCH VOL. 36

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. Ascites Adenylate Deaminase deaminase reaction. The maximal activation obtained by observed rate of adenylate depletion. The upper curve rep ADP, however, is only about 25% of that observed at the resents the more physiological situation where the 3 ade same substrate concentration using ATP as an activator. nine nucleotides are present in the reaction mixture, and High levels of ADP compete with ATP and partially reverse the response of AMP deaminase is determined as a function the ATP activation. At 1 and 2 mM ATP, a 30% reduction in of energy change variations. It is strikingly apparent from the ATP activation is observed on the addition of 10 mM Chart 5 that there is a large increase in the rate of the ADP. reaction in going from the normal physiological energy The results described above have shown that ascites AMP charge value of about 0.9 to the minimal energy charge deaminase is activated by either ATP on ADP and that the (0.55) that was observed in Chart 1 to result from addition of observed rate of the reaction depends on a complex interne glucose to intact ascites cells. The lower rates observed at lationship between the concentrations of the 3 adenine energy charge values below 0.5 are probably of no physio nucleotides. In the â€œIntroductionâ€•itwas proposed that AMP logical significance, because it is doubtful that such values deaminase functions to stabilize the adenylate energy of energy change ever occur in viable mammalian cells. charge during conditions of sudden increase in the rate of Most of the experiments reported in this study were car utilization ofATP by converting excess AMP to IMP and NH @. ned out at pH 68, whereas many of those previously re The resulting decrease in the total adenylate pool was ported were conducted in media of pH 7.2 to 7.5, which shown in Chart 1, where a 50% depletion of poo1 size was would correspond to an intracellular pH of 7.0 to 7.2 (33). seen to accompany the transient decrease (to a value of The energy charge response does not change significantly 0.55) and subsequent recovery of the energy change. over this range of pH values. In order for the AMP deaminase reaction to function in Monovalent cations are required for maximal activity. In this stabilizing capacity, it is necessary that the reaction rate Tnis-acetate buffer at pH 6.0, the saturating effect of Na@is be low or negligible at normal physiological energy charge achieved at 10 to 20 mM NaCI, with either low or saturating values (0.9) and greatly activated when the energy change concentrations of AMP. decreases. Chart 5 shows that this is the case; the concen As mentioned in the â€œIntroduction,â€•AMPdeaminase is tration of the total adenylates is held constant at 4 mM and inhibited by inorganic phosphate. Chart 6 shows the energy the energy charge is varied between 1.0 (all ATP) and 0 (all change response in the presence of phosphate at concen AMP). Since the substrate for the reaction, AMP, is one of trations ranging from 1 to 5 mM. The transient decrease in the components of the adenine nucleotide pool it is, of phosphate concentration following glucose addition (24) course, expected that the rate will increase as the energy might therefore contribute, along with the decrease in en charge decreases (and AMP increases). This is reflected in engy charge, to activating the enzyme during the initial the bottom control curve which shows the response of the phase of the experiment shown in Chart 1, as has been enzyme to increasing levels of AMP alone over the same suggested earlier by Yushok (35). range of AMP concentrations as is found in the upper curve. A series of other intermediates, both compounds known It is apparent that, in the absence of modifiers, the reaction to effect AMP deaminase from other sources and com proceeds at a very low rate even at the elevated substrate pounds whose concentration might be expected to undergo concentrations reported following glucose addition. This changes during metabolic stress, were tested as possible unstimulated rate is not rapid enough to account for the effectors. The conditions used were those of Chart 6: the energy charge response was determined between 0.6 and 0.9 at a total adenine nucleotide concentration of 4 mM in the absence and presence of various metabolites. The p0- tential effectons were present at concentrations represent ing the upper range of those encountered physiologically.

C 0

0 0.2 04 04 0.8 2 i ENERGYCHARGE Chart 5. Tbe response of ascites AMP deaminase to variations in adenyl ate energy charge at a total adenine nucleotide concentration of 4 mM. Upper set of curves, energy charge response at pH 6.8 (â€”) and pH 7.4 (- - - -); lower set of curves, response to substrate alone over the same range of AMP concentrations as is found In the upper curves. The reaction mixtures con 0.5 06 OJ 0.8 09 1.0 tamed 100 mM imidazole chloride, pH 6.8, or 100 mM Tris-CI, pH 7.4, 8 m@ ENERGY CHARGE Ng'@,0 to 4 mM AMP (lower curves), or 4 mM adenine nucleotides at energy charge values indicated. The relative amounts of ATP, ADP, and AMP at Chart 6. Inhibition of ascites AMP deaminase by inorganic phosphate. different energy charge values were calculated on the basis of a value of 0.8 The reaction mixture contained 4 m@iadenine nucleotides at energy charge for the equilibrium constant of the adenylate kinase reaction. Other condi values as indicated, 8 mM Mg'@,and 0 to 5 mM phosphate. Other conditions tions are described In the legend to Chart 2. are described in the legend to Chart 2.

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In most cases, the tests were conducted at both pH 6.8 and in energy charge to 0.5 in a 5 mM adenylate pool (AMP = 1.6 pH 7.4. Apart from inorganic phosphate, only GTP among mM), the energy charge can be restored to 0.72 in the the compounds tested was found to inhibit the reaction. At resulting 3.5 mM pool by converting 1.5 @moIesofAMP pen pH 6.8, the reaction was inhibited 30 to 50% by 0.5 mM GTP. ml to IMP. No GTP inhibition was observed at pH 7.4. The following Also, the results in Chart 7 were obtained in the absence compounds did not affect the rate of the reaction: fructose of P1and GTP. The presence of these inhibitors would cause 6-phosphate (0.2 mM), fructose diphosphate (2 mM), phos a downward shift in the family of curves. phoenolpynuvate (1 mM), lactate (10 mM), pyruvate (1 mM), citrate (1 to 2.5 mM), IMP (1 mM), glutamate (10 mM), glutamine (10 mM), DPN (0.2 mM), DPNH (0.2 mM), oxidized DISCUSSION or reduced glutathione (0.5 to 1.0 mM), and NH3(0.5 to 2.0 mM). The regulatory properties of ascites AMP deaminase sug In intact ascites cells, the glucose-induced depletion of gest that the enzyme serves to stabilize the adenylate en the adenylate pool ceases at a pool size of about 2 to 2.5 ergy charge by removing excess AMP that might otherwise mM, depending on the experimental conditions (17, 25). accumulate as a consequence of a sudden utilization of Thus, as the total adenylate concentration decreases, the ATP. rate of the reaction in vivo appears to decrease even at low The observed variation in the concentrations of the ade energy change. This is probably largely a consequence of a nine nucleotides in intact ascites tumor cells is consistent decrease in the substrate level and especially in the concen with such a proposal. Normally, both an adenylate pool level tration of ATP. The family of curves in Chart 7 represents the of 3.5 to 4 mM and a high energy charge (0.9) are maintained energy change response of AMP deaminase at different total within the cell. However, when glucose is added to aerobic concentrations of adenine nucleotides ranging from 1 to 9 ascites cells, the ensuing phosphonylation of the sugar mM. This chart shows that at an energy changevalue of 0.5 leads to a rapid decline in the cellular ATP level, as well as a to 0.6 (the minimal value attained after glucose addition) similar decrease in the level of the total adenine nucleo there is a sharp nonlinear decrease in the rate of the reac tides. When the corresponding energy charge values are tion in going from a 4 mM to a 1 mM adenine nucleotide calculated, it becomes apparent that the energy charge level. The sigmoidal response of the enzyme to ATP activa parameter is rapidly restored to control values, whereas the tion is responsible for this effect. The consequence of this replenishing of the adenylate pool is a much slower proc behavior is to prevent total exhaustion of the adenylate ess. pool. It can be estimated from the chart that when the The kinetic properties of ascites AMP deaminase meet the energy charge is 0.5 and the adenylate pool size has de necessary requirements to account for these results: the creased to approximately 1.5 mM the rate of the reaction has rate of the reaction is slow under normal physiological been reduced to that observed under control conditions conditions, it is sharply increased under conditions of low (charge, 0.9; 4 to 5 mM pool), thereby preventing or minimiz energy charge, and it remains elevated until enough AMP ing further depletion of the adenylate pool. The results has been removed to restore the energy charge to control obtained with the intact ascites cells show that the adenyl values in the diminished adenylate pool, but it decreases as ate depletion is not quite this severe. When the pool size has a consequence of the decrease in pool size. been reduced to about 2 to 2.5 mM, depending on the The observed rate of the reaction can account for the rate conditions used, depletion ceases, perhaps because this of depletion of the adenine nucleotide pool. The enzymatic degree of AMP removal is sufficient to stabilize the energy rate, expressed as nmoles/min/mg protein at 37Â°,can be change. Thus, if ATP utilization and AMP removal are in converted to @imoles/min/g packed cells by assuming a 15- balance, it can be roughly calculated that, following a drop fold purification, a 50% recovery of the enzyme activity, protein equal to one-half of the dry weight, and dry weight equal to 20% of wet weight. By these rough calculations, one finds that the rate of approximately 0.2 nmole/min/mg protein observed at low energy charge (Chart 7) come sponds to an initial rate of intracellular adenylate degnada tion of about 0.7 @mole/min/gpacked cells, or about 0.35 @mole/min/gpacked cells when an approximate 50% inhi bition by phosphate is taken into account. Lomax and Hen derson (22) found that 0.55 @moIe/gpacked cells of AMP were deaminated in the 1st 2 mm following the addition of 2- deoxyglucose to Ehrlich ascites cells, which is in close agreement with the rate estimated from the kinetic data. 0 0.2 04 U6 OLa 1.0 Both Yushok (35) and Ovengaard-Hansen (25) observed ENERGY CHARGE somewhat higher rates of adenylate degradation: 0.62 Chart 7. Response of ascites AMP deaminase to variations in adenylate @.tmoIeandabout 1 @mole/gpacked cells, respectively, of energy charge at different adenine nucleotide pool sizes. The reaction mix adenine nucleotides were degraded during the 1st mm fol tures contained 1 to 9 mM total adenine nucleotides, as specified, at energy charge values as indicated, and 1 m@ free Mg'@.Other conditions are de lowing the addition of 2-deoxyglucose or glucose to intact scribed in the legend to Chart 2. ascites cells.

1148 CANCERRESEARCHVOL. 36

Stabilization of the energy change by this method is at the [â€˜4C]adenine.Duringthe 90-mm incubation, 20% of the label expense of the adenylate concentration, which implies that was removed from the adenine nucleotide pool via the AMP it is more important for the cell to maintain a high energy deaminase reaction (10). Since IMP is an intermediate in the charge than a normal adenine nucleotide concentration. de novo synthesis of punines, the AMP deaminase reaction Farber (13) has recently reviewed several examples of nor serves no apparent biosynthetic function under normal mal physiological activity at reduced adenine nucleotide physiological conditions. Lowenstein (23) has in his punine concentrations. An adenine-requining mutant of Esche nucleotide cycle hypothesis suggested a regulatory role for richia co!i, when grown in an adenine-limiting chemostat the reaction. According to this hypothesis, AMP is deami over a 5-fold range of generation time, maintained an en nated to IMP and NH3 under conditions of high energy ergy charge that was constant within the error of the utilization (e.g., during muscle contraction), followed by a method (about 0.03), although the concentration of ATP and conversion of IMP to AMP during reduced energy demand. of total adenine nucleotides decreased by about 70% (31). It is suggested that variation in the concentrations of sub This result supports the thesis that the value of the energy strates and products of the cycle, such as NH3,modulate the charge is more important to normal cell function than is the activity of key enzymes in glycolysis and the tnicarboxylic concentration of ATP. We have not observed steady-state acidcycle. growth when the energy charge differed from its normal In certain experiments, the properties of the AMP deami value by as much as 10%. nase reaction studied here fail to account for the observed When the phosphate acceptor is a compound (like 2- changes in the different adenine nucleotide levels. For ex deoxyglucose) that cannot contribute to ATP regeneration, ample, when glyoxylate, an inhibitor of the aconitase reac the effects should be greater than those of metabolizable tion, is added to ascites cells, there is a rapid 50 to 60% sugars. Even so, it can be calculated from the results of decline in the cellular ATP level (12, 35). However, in con Lomax and Henderson (22) that the value of the energy trast to the pattern observed following glucose or deoxyglu charge was maintained at 0.5 to 0.6 in the presence of cose addition, there is only a minor change in the total deoxyglucose, while the adenine nucleotide pool decreased adenylate concentration, a 4- to 5-fold increase in the AMP by as much as 85%. This observation, like those on E. coli, concentration, and a decrease in the energy charge to a suggests that cells are programmed to avoid drastic de value of about 0.6. Since glyoxylate addition also causes a creases in energy charge, even at the expense of severe 10-fold increase in the concentration of citrate and since depletion of the adenylate pool. citrate inhibits the AMP deaminase reaction in liver, we Yushok (35) has shown that by incubating ascites cells initially assumed that the AMP accumulation was due to with adenine nucleotide precursors (adenosine on inosine) citrate inhibition of AMP deaminase. However, the enzyme the cellular adenylate pool can be augmented 1.5- to 2-fold. from ascites cells was found to be completely insensitive to It is interesting to note that the subsequent adenylate deple citrate inhibition under all conditions tested. The reason for tion resulting from addition of deoxyglucose to the cells the observed lack of AMP deamination following glyoxylate occurs at approximately the same initial rate, whether the caused ATP depletion therefore remains unclean. A reported initial pool size is 4 mM or 8 mM. This is in agreement with rise in the concentration of P1under these conditions might the results (Chart 7) that show that, at energy charge values be related to the inhibition of the enzyme activity. of 0.6 and above, the AMP deaminase reaction proceeds at The other known situation where AMP deaminase fails to nearly maximal rate at a 4 mM concentration of total ade remove accumulated AMP is when ascites cells are made nine nucleotides. anaerobic in the absence of an energy source. Aerobic If the only role of the AMP deaminase reaction is to ascites cells are able to maintain the energy change at stabilize the energy charge in the cell, then the reaction normal values for long periods even in the absence of any would be expected to be strongly inhibited under normal exogenous carbon source. In contrast, cells that have been physiological conditions. However, Charts 5 and 7 show made anaerobic can maintain a high energy charge only in that the reaction proceeds at a significant rate even at an the presence of a glycolytic intermediate. Therefore, when a energy charge value of 0.9. It can be calculated that in the suspension of starved cells is flushed with nitrogen, the presence of 4 mM adenine nucleotides and 5 mM phosphate energy charge drops rapidly to the very low values of 0.1 to these reaction rates correspond to a turnover of 1%/mm of 0.2 (12, 25, 35). The total adenine nucleotide level under the intracellular adenine nucleotide pool. Other inhibitors of goes only a slight decrease in the process, while the AMP AMP deaminase in the cell may inhibit the reaction further concentration increases severalfold. Thus there is an accu at high energy charge values. Results from experiments mulation of AMP and an apparent inhibition of the AMP with intact ascites cells indicate that the AMP deaminase deaminase reaction under conditions of low energy change reaction does normally proceed in the cell, but at a much that would appear to favor a high rate of the enzyme reac lower rate than that estimated from the kinetic data at high tion. The concentration of inorganic phosphate is appar energy charge values. Snyder and Henderson (30) report an ently higher in anaerobic ascites cells than during aerobic AMP deaminase activity of 90 nmoles/g cell/hr in intact incubation (34), and this may contribute to the inhibition of ascites cells under control conditions, which corresponds the AMP deaminase reaction. Other compounds that are to an approximate 3% turnover of the adenine nucleotide known on suspected to increase in concentration following pool per hr by the AMP deaminase reaction alone. Similar a transition to anaerobic conditions were tested as potential results were obtained earlier by Crabtree and Henderson, inhibitors of the reaction, but with negative results. who incubated ascites cells with low concentrations of A dense population of ascites cells growing in vivo might

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. A. G. Chapman et al. encounter reduced oxygen tension, but the cells probably 1086, 1971. 10. Crabtree, G. W. C., and Henderson, J. F. Pathways of Purine Ribonucleo share the ability of other mammalian cells to withstand very tide Catabolism in Ehrlich Ascites Tumor Ceilsin Vitro. Can. J. Biochem., low oxygen tension without any change in the concentra 49:959-963,1971. tions of the adenine nucleotides. Only at partial pressures of 11. Criss, W. E. Control of the Adenylate Charge in the Morris â€œMinimal Deviationâ€•Hepatomas. Cancer Res., 33: 51-56, 1973. oxygen below 18 mm Hg is there a decrease in the energy 12. Criss, W. E. Control of the Adenylate Charge in Novikoff Ascites Cells. change value of brain (29) or liver (5). Thus the reason for Cancer Res., 33: 57â€”64,1973. apparent inhibition of AMP deaminase in ascites cells under 13. Farber, E. ATP and Cell Integrity. Federation Proc., 32: 1534-1539, 1973. 14. Faupel, R. P., Seitz, H. J., Tarnowski, W., Thiemann, V., and Weiss, C. The an atmosphere of 100% nitrogen remains obscure, but it is Problem of Tissue Sampling from Experimental Animals with Respect to unlikely thatit is of physiological significance. It has also Freezing Technique, Anoxia, Stress and Narcosis. Arch. Biochem. Bio phys., 148: 509â€”522,1972. recently been shown that there is a rapid and immediate 15. Fox, I. H. Purine Ribonucleotide Catabolism: Clinical and Biochemical decrease in viability of starved ascites cells when incubated Significance. Nutr. Metab., 16: 65-78, 1974. at pH 7.35 under anaerobic conditions (26). 16. GUnther, T., and Dorn, F. Die Intrazellulare Mg-Ionenaktivitat in Verschi denen Saugetierzellen. Z. Naturforsch., 26b: 176-1 77, 1971. It has been suggested that ascites tumor cells might have 17. Gurd, J. W., and Scholefield, P. 6. The Energy Metabolism of Novikoff somewhat higher energy charge values than nonmalignant Ascites Hepatoma Cells. I. The Effects of Glucose on the Intracellular cells such as liven (11, 20). Part of the apparent difference is Level of Adenine Nucleotides. Can J. Biochem., 49: 686â€”694,1971. 18. Ibsen, K. H., and Schiller, K. W. Control of Glycolysis and Respiration in probably due to the greater difficulty, as compared with a Substrate-Depleted Ehrlich Ascites Tumor Cells. Arch. Biochem. Bio suspension of incubated ascites cells, of sampling an intact phys., 143: 187-203, 1971. 19. Kalckar, H. N. Differential Spectrophotometry of Purine Compounds by tissue sufficiently rapidly. Liven data obtained under cane Means of Specific Enzymes.Ill. Studies of the Enzymesof Purine Metab fully controlled conditions show energy change values very olism. J. Biol. Chem., 167: 461-475, 1947. similar to those seen in tumor cells (0.90 to 0.92) (7, 14). 20. Keppler, D. 0. R., and Smith, D. F. Nucleotide Content of Ascites Hepa toma Cells and Their Changes Induced by o-Galactosamine. Cancer Although the malignant cells appear to rely more heavily on Res., 34: 705-711, 1974. their cytoplasmic enzyme system for regenerating ATP (11, 21. Lee, I. Y., Strunk, R. C., and Coe, E. L. Coordination among Rate-limiting 12), the concentrations of the adenine nucleotides and the Steps of Glycolysis and Respiration in Intact Ascites Tumor Cells. J. Biol. Chem., 242: 2021-2028, 1967. energy change values are very similar in malignant and 22. Lomax, C. A., and Henderson, J. F. Adenosine Formation and Metabolism nonmalignant cells. Furthermore, liven and ascites cells me during Adenosine Triphosphate Catabolism in Ehrlich Ascites Tumor Cells. Cancer Res., 33: 2825-2829, 1973. spond in a similar manner to sudden energy demand in the 23. Lowenstein, J. M. Production in Muscle and Other Tissues: The Purine form of addition of high concentrations of phosphate-ac Nucleotide Cycle. Physiol. 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Regulatory Mechanisms in Carbo hydrate Metabolism. IX. Stimulation of Aerobic Glycolysis by Energy 1. Atkinson, D. E. The Energy Charge of the Adenylate Pool as a Regulatory linked Ion Transport and Inhibition by Dextran Sulfate. J. Biol. Chem., Parameter. Interaction with Feedback Modifiers. Biochemistry, 7: 4030- 248: 5175â€”5182,1973. 4034, 1968. 28. Seligson, D., and Seligson, H. A Microdiffusion Method for the Determi 2. Atkinson, D. E. Adenine Nucleotides as Stoichiometric Coupling Agents nation of Nitrogen Liberated as Ammonia. J. Lab. Clin. Med., 38: 324-330, in Metabolism and as Regulatory Modifiers. In: H. J. Vogel (ed), Meta 1951. bolic Regulation, pp. 1-21. New York: Academic Press, Inc., 1971. 29. Siesjo, B. K., and Nilsson, L. The Influence of Arterial Hypoxemia upon 3. Atkinson, D. E. The Adenylate Energy Charge in Metabolic Regulation. In: Labile Phosphates and upon Extracellular and Intracellular Lactate and A. San Pietro and H. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. Role of the Adenylate Deaminase Reaction in Regulation of Adenine Nucleotide Metabolism in Ehrlich Ascites Tumor Cells

Astrid G. Chapman, Arnold L. Miller and Daniel E. Atkinson

Cancer Res 1976;36:1144-1150.

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