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

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The Hydrolysis of ATP and Related Nucleotides by Ehrlich Ascites Carcinoma Cells* 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 5—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 ULLREY—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 1—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.—In 924 experiments intact equivalent to 10@cells and 106 mg. of dry weight. Ehrlich ascites carcinoma cells produced 90 ±9292 Cells were incubated at 37°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 (0.1— 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 11.2±5.2 0.4±0.5 cell and substrate blanks were also included. Dis Mitochondria 28.9±7.8 5.3 ±2.5 Microsomes 56.3±2.8 19.8±5.9 appearance of ATP from cell supernatant fluids Supernatant 7.6±2.7 25.6±4.0 was also measured directly by the method of @:100 104 ±8.3100 51.1±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 ±92percent variation in any onebatch of cells. P1 from ATP or ADP by whole homogenates. Homogenates and fractions incubated at 37°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 ±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, Mg±+ 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 4°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. Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1962 American Association for Cancer Research. 9230 Cancer Research Vol. 9292,February 19692 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 37°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 ±0.4 and 6.4 ± TABLE 2 0.6 jzmoles/cc of cells, respectively.
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