The Hydrolysis of ATP and Related Nucleotides by Ehrlich Ascites Carcinoma Cells*

DONALD F. HOELZL WALLACH AND DONNA ULLREY

(Department of Biological Chemistry, Harvard Medical School, and Protein Foundation, Boston, Mass.)

SUMMARY The ATPase of Ehrlich ascites carcinoma cells has been examined. The evidence presented indicates the following: (a) ATP hydrolysis occurs at the cell surface, (b) the cell membrane goes into the microsomal fraction upon cell rupture and differ ential centrifugation, (c) ATP hydrolysis depends upon the nature and concentration of external activator ions, (d) hydrolysis is not limited to ATP; other nucleoside tn phosphates are attacked with equal vigor, and ADP and AMP are also hydrolyzed, (e) ATP hydrolysis appears to proceed in stepwise fashion via ADP and AMP to ade nine and inorganic phosphorus, (J) ATP, but not ADP or ITP hydrolysis, is stimulated by 92,4-dinitrophenol, but this effect is reversed by glucose.

The presence on the surface of a variety of phate by Ehrlich ascites carcinoma cells was first cells of enzymes capable of hydrolyzing adenosine recorded by Acs et at. (92). triphosphate has been reported by several workers. The above studies suggest the possibility that Rothstein et al have presented evidence for the adenosine tniphosphate-hydrolyzing enzymes may localization of an adenosine tniphosphatase on the exist as an integral part of the lipoprotein matrix surface of yeast (920) and of intestinal epithelium of cell membranes. In the course of examining (921). Libet (13) and Abood et a! (1) have shown this hypothesis for the case of the Ehrlich ascites that the adenosine triphosphatase of the giant carcinoma, we have studied in some detail the axon of the squid lies in the axon sheath rather cleavage of adenosine triphosphate and related than in the axoplasm. Essner et at. (9) have pre substances by these cells. sented evidence localizing the adenosine triphos MATERIALS AND METHODS phatase of liver cells in the plasma membrane of the microvilli of bile cannaliculi ; Spater et The tumor used was the hyperdiploid Ehrlich at. (924), also using cytochemical methods, have ascites carcinoma carried by weekly transfers in demonstrated adenosine triphosphatase activity Swiss white mice. Tumors 5â€”8days old and free in the plasma membranes of proximal renal tubular of erythrocytes were used. For isolation of the cells of a variety of species; and Novikoff (15, 16) cells 10 cc. of 0.15 N sodium chloride was injected has shown the presence of ATPase on the surface into the peritoneal cavity of the host, and the of a variety of tumor cells. resulting cell suspension was withdrawn. The cells The stromal adenosine tniphosphatase of eryth were washed twice with 10 volumes of 0.15 N rocytes has been the subject of considerable sodium chloride and were suspended to a concen study (6, 8, 11, 927, 392). It has recently been tration of 10 per cent in the medium composed implicated in the cation transport mechanisms as follows: NaCl, 0.1492 M; KC1, 0.005 M; tris of these cells by Post et at. (18). A potent adenosine (hydroxymethyl)aminomethane, 0.037 M; pH 7.40. triphosphatase on the surface of human leukocytes Orthophosphate in the form of a sodium phos has been reported by Luganova et at. (14). The phate buffer, pH 7.4, was added to the above in hydrolysis of externally added adenosine triphos appropriate amounts when the effects of phos phate ion were to be studied. The osmolanity C This work was supported by a grant from the National of the medium was then adjusted by omitting Institutes of Health (C-3943). the appropriate amount of sodium chloride. Received for publication August 21, 1961. After a stock cell suspension of about 10 per 92928

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1962 American Association for Cancer Research. WIALLACH AND ULLREYâ€”Jfydrolysis of 92929 cent had been prepared, a precise packed cell (928).Air-dried, unfixed smearswereused,since volume was obtained by centrifuging an aliquot fixation in cold calcium-formol (3) led to marked in a Wintrobe hematocrit tube at 4500 X g for inactivation of the ATPase. 45 minutes. Other aliquots of the stock suspension were diluted to give final cell concentrations of RESULTS 1â€”3 mg of cells per ml. of suspension. As de As8ociation of the A TPa@e with sedimentable termined previously (30) 1 cc. packed cells is components of the celi.â€”In 924 experiments intact equivalent to 10@cells and 106 mg. of dry weight. Ehrlich ascites carcinoma cells produced 90 Â±9292 Cells were incubated at 37Â°C. with gentle shaking @molesinorganic phosphorus/mi cells/hr from ex for periods of .5minutes to 92hours. All experiments ternally added ATP(0.5 nmz, initial concentration). were carried out under aerobic conditions. Sub The ATPase activity of homogenates was 92.5 strate concentrations ranged between 0.05 and times that of intact cells under identical condi @5.0 m@. Nucleotides used were obtained from tions. The distribution of ATPase activity in the Pabst and Co.' After completion of incubation subcellular fractions is shown in Table 1. Ninety the cells were chilled, removed by centrifugation, two per cent of the ATPase activity was associated and the supernatant fluid was analyzed for inorganic with sedimentable subcellular fractions, most of phosphorus (10) and 11-minute hydrolyzable phos TABLE 1 phorus (4, 7) after deproteinization with 10 per cent trichloroacetic acid. DIsTRIBuTIoN OF ATPASE AND ADPASE AcTwrrY iN Because of the very low cell concentration (0.1â€” SUBCELLULAR FRACTIONS FROM EimLIcH 0.3 per cent by volume), more reliable data could ASCITES CARCINOMA CELLs be obtained by deproteiizing the chilled cell suspension directly, without prior removal of cells. ACTIVITYATPADPHomogenate In the bulk of the reported experiments, other FRACTIONENZYME than those where both extra- and intracellular labile and inorganic phosphorus were measured, this technic was employed but the appropriate Nuclei 11.2Â±5.2 0.4Â±0.5 cell and substrate blanks were also included. Dis Mitochondria 28.9Â±7.8 5.3 Â±2.5 Microsomes 56.3Â±2.8 19.8Â±5.9 appearance of ATP from cell supernatant fluids Supernatant 7.6Â±2.7 25.6Â±4.0 was also measured directly by the method of @:100 104 Â±8.3100 51.1Â±5.6 Strehler and Trotter (925). Determinations were carried out in triplicate and showed no more than Enzyme activity expressed relative to the rate of release of Â±92percent variation in any onebatch of cells. P1 from ATP or ADP by whole homogenates. Homogenates and fractions incubated at 37Â°C. for 1 hour in 0.25 M sucrose Whereas the ATPase activity was highly repro containing Mg@ (0.5 mM) and CN (1.0 mM). initial substrate ducible in any one tumor, it varied by Â±924 concentration 0.5 mM. Data show means and S.D.'s of three per cent from tumor to tumor. Because of this, separate experiments. in all experiments the rate of production of inor ganic phosphorus from ATP (0.5 m@ initial con it in the microsomes. In contrast, most of the centration, MgÂ±+ 0.5 m@) was included as an ADPase activity was found in the microsomal and internal standard. soluble fractions. For preparation of homogenates 10 per cent The microsomal ATPase is associated with the cell suspension in 0.925M sucrose + .00092M MgCl2 microsomal membranes, which can be readily sepa were subjected to 800 lb. N2 per sq. in. for 920 rated from the ribosomes by differential centrif minutes at 4Â°C. followed by a sudden return to ugation. Thus, if whole microsomes are centni atmospheric pressure. Under the above conditions fuged for 18 hours at 105,000 X g in a sucrose cell rupture, which occurs due to nitrogen cavita gradient ranging from 492 to 50 per cent, 892 per tion in the cytoplasm, is quantitative (192).Homog cent of the ATPase and 78 per cent of the ADPase enates were separated into the various subcellular are found in the lighter half of the gradient, which fractions as reported previously (30). also contains 77 per cent of the phospholipide Histochemical demonstration of ATPase was but only 17 per cent of the RNA. The membrane performed by the method of Wachstein and Meisel preparation thus obtained can be further separated

1 The following abbreviations are used: AMP = adenosine by centrifugal means into at least three distinct 5'-monophosphate; ADP = adenosine diphosphate; ATP fractions, all of them free of RNA.2 adenosine triphosphate; CTP = cytidine triphosphate, Experiments on the effects of RNase upon GTP = guanosine triphosphate; UTP = uridine triphos microsomal ATPase support the above observa phate; Pi = inorganic phosphate; DNP = 2,4-dinitrophenol; RNA = ribonucleic acid; RNase = ribonuclease. 2 Donald F. H. Wallach, to be published.

tions. Thus incubation of a dilute microsomal activity was undiminished. There was also no suspension with 0-0.092 per cent RNase at pH leakage of ADPase into the medium. 7.4 and 37Â°C. for 1 hour led to a loss of only Experiments were also performed to check 16 per cent of ATPase activity. whether ATP addition to the cell suspension me When RNase-treated microsomes were centri dium influenced the intracellular labile and inor fuged at 105,000 X g for 30 minutes no ATPase ganic phosphate levels, but no significant effect was found in the supernatant, and less than 50 was found. Thus, the average levels of intracellular per cent of the original activity could be recovered inorganic phosphorus and acid-labile phosphorus in the absence of ATP were 10.6 Â±0.4 and 6.4 Â± TABLE 2 0.6 jzmoles/cc of cells, respectively. In the presence HYDROLYSIS OF AT? AND RELATED of ATP in the suspension medium at a concentra SUBSTANCES BY Emtucu ASCITES tion of 1 nmi, the levels were 11.3 Â±0.4 and 5.6 Â± CARcINoMA CELLS 0.3, respectively. Type. of phosphate esters attacked by Ehrlich SubstrateHydrolysi.*ATP100ADP ascites carcinoma cell, and path. of hydrolyai..â€”As shown in Table 92,all the nucleoside triphosphates 9 were hydrolyzed with relatively little preference. AMP73Â±(4)C,rP89(1GTP 92Â±10(5) Inorgani@ pyrophosphate, ADP, and AMP were also vigorously attacked, but @-glycerophosphate ITP 85Â±14 (4) was split at only a very feeble rate. UTP 89 (1) As might be expected from these data, both P207 74Â±24 (3) $-Glycerophosphate100 13 Â± 5(1 (2) of the phosphate anyhdride linkages and the phos phate ester group of ATP are attacked by the

* Hydrolysis rates expressed relative to the rate of release of P@from AlP at an initial concentration of 0.5 mM. All nu @&M0t.ES cleotides present at initial concentration P'1 of 0.5 mM. Activating ion = Mg@ (0.5 cc C&ISU$pL mM). Cells incubated in standard medi P0 urn. Data show means and S.D.'s for the number of experiments given in paren theses.

in the pellet. This loss of ATPase activity in RNase-treated microsomal pellets has been re ported previously by the authors (81) and is probably due to the aggregation of microsomal vesicles following RNase treatment, which makes resuspension of the pellets very difficult. The ATPase of Ehrlich ascites carcinoma cells is readily demonstrable histochemically by the PH-P0 lead-nucleotide method of Wacb.stein and Meisel (928). As reported for other ascites cells by Novi TIME IN MINUTES koff (17), the ATPase activity of Ehrlich ascites CHART 1.â€”Hydrolysis of ATP by Ehrlich ascites carcinoma carcinoma cells is associated with the cell surface cells. Incubation in standard medium, with Mg@ (10@ w) and is demonstrable after 15 minutes of incubation used as activating ion. Initial ATP concentration, 1.25 m@. at 37Â°C. On the other hand, no ADPase activity Cell concentration, S mg/cc. P,, = 11-minute hydrolyzable could be demonstrated histochemically even after phosphorus. P0 = inorganic and highly labile phosphorus. 1920 minutes, and hydrolysis of AMP appeared to be restricted to the nuclei and could be detected Ehrlich ascites carcinoma cell. This is illustrated only after 60 minutes.2 in Chart 1, which shows the linear disappearance To test whether any ATPase leaked from the of 11-minute hydrolyzable phosphorus and ap cells during incubation, as is the case with some pearance of inorganic phosphorus with time, the enzymes (5), cell suspensions were gently shaken latter being greater than the loss of labile phos aerobically for 1 hour at 37Â°C., and the cells were phorus. then removed by centrifugation. No ATPase ac It seems probable that a single enzyme attacks tivity was found in the cell supernatant, and cell both ATP and II?, since the rate of hydrolysis

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1962 American Association for Cancer Research. WALLACH AND ULLREYâ€”Hydrolysis of Nucleotides 9231 of ATP (.5 nm&) in the presence of ITP (.5 mM) of ATP and ADP (log K1 for Mg-ATP = 3.47@ was only 37 per cent of that found with ATP and for Mg-ADP = 3.00, where K1 are the asso alone. In any case, it appears that the two phos ciation constants) (923). This does not occur in phate anhydride linkages are broken sequentially, the case of AMP, probably because of the low followed by hydrolysis of the resulting phospho affinity of AMP for Mg++ (log K = 1.69). monoester. This contention is supported by the The rate of hydrolysis of these substrates varied inhibition exerted by ADP and AMP on ATP linearly with the amount of cellular material pres hydrolysis and of AMP on ADP hydrolysis (Ta ent between 0.05 and 1.925 mg cells/ml cell sus bIeS). pension. When the disappearance of ATP was followed Ion dependence.â€”Contrary to the report by by the luciferase technic, the rate of hydrolysis Acs et at., we found that the surface ATPase of ATP (0.5 nm@, initial concentration) in the of Ehrlich ascites carcinoma cells is markedly presence of an equimolar concentration of ADP dependent upon the presence of externally added was 85 per cent of that found in the absence TABLE 3 of ADP. At the same time, the rate of production INTERRELATIONSHIPS BETWEEN THE HYDROLYSIS OF of inorganic phosphorus was 892per cent of that AlT, ADP, AND AMP BY ERHLICH from ATP alone and only 51 per cent of that ASCITESCARCINOMACEus expected if the hydrolysis of ATP and ADP were independent. Interestingly enough, an identical pattern could be demonstrated with ITP and ADP. These data could be explained by assuming [email protected] that ATP and ADP are attacked by a single en mMATP mMMg@. 1.0 zyme; but this seems improbable in view of the differing activator ion needs for ATP and for ADP ADP 61 78 AMP 96 96 hydrolysis. It is more probable that the restriction of ATP hydrolysis by ADP is due to product fadditive 161 192 ATP+ADP 1@found inhibition. In either case our data are not consist 82 84 ent with the possibility that ADP breaks down fadditive 196 210 ATP+AMP @found via an adenylatekinase pathway. 116 113 Similar interrelationships can be shown to exist Jadditive 157 174 between ATP and AMP as well as ADP and AMP ADP+AMP @found100 58114 63 (Table 3). Thus, the inorganic phosphorus released was only 59 per cent of the expected value when * Hydrolysis rates expressed relative to the P, release from ATP and AMP were both present at a concen Al?, which was used as reference for each series. All substrates at05 mM initialconcentration,with Mg@asactivatorat stated tration of .5 mM and only 37 per cent in the case levels. Incubations in standard medium for 1 hour at 87Â°C. of ADP and AMP (.5 m@i).Even greater inhibition Data are from two cell pools, one for each Mg@ concentra was found at the higher magnesium concentration, tion. Experiments with initial substrate concentrations of 0.2 mM gave equivalent results. which excludes the possibility that those effects were due to magnesium binding by the nucleotides. divalent cations (Table 4). The activity without In contrast to the above interdependence of the divalent cations was minimal. At the ion con hydrolysis of ATP, ADP, and AMP, no significant centrations illustrated here (5 X 10@i) maximum effect of @-glycophosphate on AMP hydrolysis activation occurred with Ca@. Mg@ was only could be shown, nor was there any effect of in slightly less effective, Mn@ considerably less, and organic phosphate (1â€”10nmt) upon ATP hydrol Co@ least so. Optimum activation occurred at ysis. concentrations of 10@t for both calcium and The effect of substrate and cell concentroiion upon magnesium. At this concentration also, Ca@ was the rate ofhydrolysis ofA TP, ADP, and AMP.â€”AS slightly more effective than Mg@. No competition shown in Chart 92for two magnesium concentra between Ca@ and Mg@ was found; on the con tions, the rates of hydrolysis of ATP, ADP, and trary, their effects were additive. Thus, the ac AMP at low initial concentrations appear to fol tivity obtained in the presence of 5 X 1O@Mcal low first-order kinetics. The strong dependence cium plus 5 X 10â€”4MMg@ was equivalent to the of activity on Mg4-'- concentration at high con activity obtained in the presence of 10@ Mg@. centrations of ATP and ADP, and particularly As might be expected the ATPase was completely the inhibition of activity at high@ATP levels, is inhibited by EDTA (10@i) and by fluoride ion best attributed to the MgH@complexing action (102M).

In contrast to the marked ion dependence of the dinitrophenol (10@ to 5 X 10@i) without glu ATPase, adenosine diphosphatase activity was cose increased ATPase activity by 920â€”47percent. much less sensitive to externally added cations At lower concentrations of this inhibitor (1O@ to (Table 4). Nevertheless, definite stimulation was 10@i) no effect was observed. Of particular inter obtained with Ca@, Mg++, and Mn@, but not est is the fact that dinitrophenol gave an identical Co@. Maximal rates were again obtained at con increment in production of inorganic phosphorus centrations of Ca@ and Mg+@ of 10@, but these when the experiment was carried out without were only about 30 per cent greater than without externally added activator ion. added activator cations. However EDTA (10@z) CN (10@i) produced a similar but less marked fully inhibited ADPase activity, and the ADPases effect. The stimulation of ATPase by dinitrophenol of the microsomes and soluble supernatant were and by cyanide was more than completely reversed entirely inactive without added activator ions. by the addition of glucose at concentrations of The effect of glucose and of metabolic inhibitors.â€” about 10-2M. Thus, whereas dinitrophenol in the The presence of glucose (10@i@) slightly, but con absence of glucose produced a 19 per cent stimu sistently, reduced ATP hydrolysis by Ehrlich as lation of ATPase activity, the rate of hydrolysis cites carcinoma cells (Table 5). On the other hand, of ATP in the presence of 10@ dinitrophenol

140

@..% 00 U) -J w-J 0 -j 60

@:40 0@ (I) Ui -.J 0 0 0

NUC'LEOTIDE CONCENTRATION (p@M)

CHART 2.â€”Effect of substrate concentration upon the hy cells. Incubation for 1 hour at 37Â°C., with Mg@ used as drolysis of ATP, ADP, and AMP by Ehrlich ascites carcinoma activator ion at stated concentrations.

TABLE 4 THE EFFECT OF DIVALENT CATIONS UPON TABLE 5 THE HYDROLYSIS OF Al? AND ADP BY THE EFFECT OF 2,4-DINITROPHEN0L EHRLICH ASCITES CARCINOMA CELLs AND GLUCOSE UPON AlP HYDROL YSIS BY EIIRLICH ASCITEs C@aci NOMA CELLS DIVALENT CATIONHrirnoLYsIs5AlPADPMg@ hydrolysis5ControlConditionsAlP

Ca'@ 112 (Â± 4) 117 (Â±11) DNP 131 (Â±7) Mn@@ 88 (Â±8) 91(Â±0 Glucose 90(Â±6) Co@ 54 (Â± 3) 65(Â± 5 DNP+glucose100 93 (Â±3) None100 2 .5 (Â±.5)100 72 (Â± 6)

S Hydrolysis rates expressed relative to * Hydrolysis rates expressed relative to the the rate of release of P, from AlP at an rate of release of P, from ATP and ADP in the initial concentration of 0.5 mM in the presence of Mg@asactivating ion.ATPand ADP presence of Mg@ (0.5 mM). DNP con present at an initialconcentration of 0.5 mM. Cat centration, 0.1 mM. Glucose concentra ion concentrations, 0@5mM. Cells incubated in tion, 10 mM. Cells incubated in standard standard medium for 1 hour at 37Â°C. Data medium for 1 hour at 37Â°C.Data show show means and S.t).'s of three experiments. means and S.D.'s of three experiments.

and 102M glucose was identical to that in the these cells, is the finding that most of the nonmito presence of glucose alone. Unlike the effect of chondrial ATPase is in the microsomal mem dinitrophenol on ATP hydrolysis, no effect of branes. There is growing evidence that the plasma this agent on the hydrolysis of ADP or ITP membranes of a variety of cells form small frag could be demonstrated. ments upon cell rupture which sediment with the microsomes after differential centrifugation. Thus, DISCUSSION Novikoff has shown that, upon cell rupture, the Clearly, Ehrlich ascites carcinoma cells can surface membranes of both a solid (16) and an hydrolyze externally added ATP as well as a va ascites hepatoma (17) formed small vesicles not riety of other phosphate esters. Two important unlike the vesicles of the endoplasmic reticulum. questions arise in this regard : The first is whether The transplantation antigenicity of microsomes the ATPase of these cells is on or a part of the from the Novikoff hepatoma (16) and the human surface membrane; the second is what the function tumor H.Ep. #3 (926)have also been ascribed to the of this and its associated enzyme systems might be. presence of cell membrane fragments in the micro As far as the localization of these enzymes is somes. Finally, Wallach and Eylar (929) have re concerned, there are really only two possibilities: cently found that the negative surface charge First, hydrolysis occurs within the cellâ€”i.e., the of Ehrlich ascites carcinoma cells is due largely substrates diffuse into the cell, are cleaved, and to sialic acid and that most of the sialic acid of the products of hydrolysis diffuse out again. This these cells is found in microsomal membranes suggestion, which has already been discussed for following cell rupture. These studies again suggest yeast by Rothstein (920), appears highly improb that the surface membrane either sediments with able here also. Certainly, mammalian cells are the microsomal membranes after pressure homog not readily permeable to polyphosphates such as enization or that it is continuous with them in the nucleotides under discussion, and even inor the intact cell. ganic phosphorus moves into Ehrlich ascites car The function of cell surface ATPases is as yet cinoma cells not by diffusion but by an active unclear. Certainly the studies of Dunham (8), process (19). In any event, the problem is even Whittam (392), and Post et at. implicate erythro more complex, since the cleavage sites are not in cyte ATPase in cation transport. To date, our the soluble proteins underlying the surface mem studies have not shown any definite effect upon brane but in the microsomes and mitochondria. ATPase activity of intact Ehrlich ascites cells Even if it is assumed that the nucleotides can other than that produced by the action of dinitro enter the cell and are hydrolyzed there, the prob phenol and glucose. It appears that, in common lem of the movement of inorganic phosphorus with mitochondria, the surface ATPase is stimu from the cell remains. The present data show that lated by dinitrophenol. However, in the latter externally added ATP does not affect internal case the stimulation is fully reversed by glucose. ATP or inorganic phosphorus levels, yet inorganic These data suggest that the activity of the surface phosphorus moves out of cells only very slowly2 ATPase in some way reflects intracellular energy even when external levels are zero and internal requirements. Particularly cogent to this is the levels are raised artificially by DNP (9292). fact that DNP had no effect upon ITP or ADP The second possibility is that cleavage of ATP cleavage. occurs on the cell surface. Apart from the above In any event it appears probable that cell considerations, three factors support this conten surface processes such as active transport, ameboid tion. The first is the histochemical localization motion, and pinocytosis utilize ATP as an energy of ATPase on the cell surface of the Ehrlich ascites source and hence may act as â€œATPases.â€•Possibly carcinoma as well as other cells. The second is these mechanisms are not rigidly oriented toward the striking dependence of ATPase activity upon the cell interior and can thus utilize external ATP. the nature and concentration of external activator Unfortunately, there is no information whether ions. In this connection it is aLso of interest that the energy yielded by surface ATP hydrolysis ADPase activity in these cells, which lies to a is actually used to drive cellular endergonic proc large extent in the 105,000 X g supernatant and esses. It is also unknown what function is served which is relatively independent upon external ac by ADP and AMP hydrolysis, but our data may tivator ion concentration, cannot be visualized indicate that the ADPase is not a membrane histochemically. bound enzyme but is a soluble protein which A third factor, consistent with the localization becomes trapped in the microsomal vesicles during of ATPase activity on the surface membrane of homogenization.

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Donald F. Hoelzl Wallach and Donna Ullrey

Cancer Res 1962;22:228-234.

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