Proc. Natl. Acad. Sci. USA Vol. 73, No. 9, pp. 3122-3125, September 1976 Biochemistry
Incorporation of adenosine into ATP: Formation of compartmentalized ATP (radioactive labeling in vivo/adenine nucleotides/tumors)
ELIEZER RAPAPORT AND PAUL C. ZAMECNIK The John Collins Warren Laboratories of the Huntington Memorial Hospital of Harvard University at the Massachusetts General Hospital, Boston, Mass. 02114
Contributed by Paul C. Zamecnik, June 28, 1976
ABSTRACT The incorporation of [3H]adenosine, [3H]adenine, and [3Hlhypoxanthine into adenine nucleotides of nude (athymic) mouse liver and human hepatoma grown subcutaneously in nude mice was studied. 3H and 32P radioactive labeling in vivo of acid-soluble nucleotides followed by chromaprocedures indicated that, in contrast to [3Hjadenine tographic and [ H]hypoxanthine, [3H]adenosine is preferentially incorporated into ATP in comparison with its incorporation into AMP and ADP. This phenomenon, as well as complementing the recently reported 3fold increase in total cellular ATP upon treatment with 0.5-1.0 mM concentrations of adenosine, indicates the formation from adenosine of compartmentalized ATP that is not produced from either adenine or hypoxanthine. The observed effect is of larger magnitude in the growth-arrested normal liver than in the actively growing tumor.
MATERIALS AND METHODS Nude (athymnic) mice were obtained from Dr. WendallFarrow of Life Sciences Research Laboratories, through the auspices of the Office of Program Resources of the Virus Cancer Program, National Cancer Institute. A human malignant hepatoma cell line was developed from a primary tumor by Dr. Kurt Isselbacher's laboratory. The cell line is malignant, as judged by progressive infiltration and lethality in the nude mice, and is capable of synthesizing certain proteins characteristic of human liver. About 5 X 107 cells were injected subcutaneously into nude mice (16-19 g). After 10-14 days a solid tumor of 0.5-1.0 g developed and mice were used at this stage. Radioactive chemicals were supplied by New England Nuclear (Boston, Mass.). 32P1 (0.1 mCi in 0.9% NaCl) was injected intraperitoneally, and 3H-labeled precursors (0.5 mCi in 0.9% NaCl) were injected intravenously in a total volume of 0.2 ml. All 3H-labeled compounds were of the highest specific activity available (15-25 Ci/mmol). These procedures as well as the final dissection and freeze-clamping of liver or tumor were carried out under ether anesthesia in an atmosphere of 100% oxygen to minimize hypoxia (10). Livers and tumors were excised and within 1-5 sec were freeze-clamped between heavy aluminum plates precooled in liquid nitrogen (11). The frozen tissue was pulverized in a mortar at solid CO2 temperature and extracted for at least 30 min with ice-cold 0.5 M perchloric acid. Following removal of cell debris by centrifugation, the solution was neutralized with 7 M KOH. Two procedures were used for analyzing acid-soluble nucleotides, and both gave similar results. Acid-soluble nucleotides were analyzed on a DEAESephadex A-25 column, eluted with a linear gradient of 0.1-0.6 M NaCl in 0.05 M Tris-HCl, 0.002 M EDTA, and 7 M urea, pH 7.6. This procedure affords a separation according to increasing negative charge. The fractions containing ADP and ATP were well separated. The fractions containing AMP (as well as those containing ADP and ATP) were diluted 5-fold and applied on top of a Dowex 1-X8 (formate) column. Elution with a linear gradient of 0.0-2.0 M ammonium formate, pH 5.0, afforded further purification of these nucleotides. The second sequence which was utilized more often included initial chromatography of acid-soluble nucleotides on a DEAE-cellulose column eluted with a linear gradient of 0.0-0.5 M ammonium bicarbonate, pH 8.1. Appropriate fractions were pooled and the buffer was removed by repeated evaporation and lyophilization. The dry residues were dissolved in a small volume of water and part of the solution was subjected to snake venom phosphodiesterase digestion followed by thin-layer chromatography on PEI-cellulose. Elution was performed with water followed by 1 M LiCl. The plate was cut and the small pieces were eluted with 4 M ammonium hydroxide for at least 1 hr, before measuring the
Several recent reports have indicated that treatment of different tissues with 0.5-1.0 mM concentrations of adenosine caused a marked increase in their ATP levels (1-5). It was shown that this increase, which was also reflected in high ATP/ADP ratios, could not be attained with other adenine nucleotide precursors, namely, adenine, hypoxanthine, or inosine (3). Adenosine has been found to be lethal to cells in culture at a low concentration (6), as the result of a metabolic imbalance which shuts off pyrimidine biosynthesis. This lethality was abolished in mutants which did not contain adenosine kinase and was thus attributed to a phosphorylated intermediate (6). The reported stimulatory effect of small adenosine concentrations on DNA synthesis in the presence of serum (7) can be attributed at least in part to inosine formed from adenosine with adenosine deaminase present in serum in tissue cultures (Rapaport and Zamecnik,
unpublished data). Adenosine kinase and adenosine deaminase
are the enzymes known to metabolize adenosine in mammalian tissue (8), yielding AMP and inosine, respectively. Inosine is an established intermediate in the purine salvage pathway (9). We have utilized high specific activity radioactive precursors to study the incorporation of adenosine into ATP in nude mouse
liver and subcutaneous, fast-growing human hepatoma in the nude mouse. The results indicate that in both tissues [3H] adenosine but not [3H]adenine or [3H]hypoxanthine is preferentially incorporated into ATP relative to its incorporation into AMP or ADP. Thus, the existence of a pathway which leads to a compartmentalized ATP pool from adenosine is indicated. This phenomenon is of a larger magnitude in the liver than in the hepatoma grown in the same nude mouse. Polyomatransformed baby hamster kidney cells (BHK21/C13) grown in the nude mouse yielded similar results. The direct comparison of the total ATP levels and the metabolically significant ATP/ADP ratios in liver and tumor of the same live animal, as well as the different magnitudes of ATP
compartmentalization in the liver and tumor, have been studied.
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FIG. 1. DEAE-Sephadex-7 M urea column chromatography of acid-soluble nucleotides from nude mouse liver (a) and tumor (b). Radioactive labeling was performed with 0.1 mCi of 32Pj for 2 hr followed by 0.5 mCi of [3H]adenosine for 30 min. The tumor was excised first; hypoxia caused by some bleeding is the probable reason for the low ATP/ADP ratio in the liver.
FIG. 3. DEAE-cellulose column chromatography of acid-soluble nucleotides from nude mouse liver (a) and tumor (b). Radioactive labeling was performed as described in the legend to Fig. 1 except that [3H]adenosine was injected for a period of 20 min before excision of the liver, followed by removal of the tumor.
radioactivity of the ammonium hydroxide eluate in Bray's scintillation fluid. Snake venom phosphodiesterase digestions were performed in 0.05 M Tris-HCl, 0.05 M MgCl2, pH 8.4, at 300 for 1 hr.
analyzing the acid-soluble nucleotides. DEAE-Sephadex-7 M urea column chromatography, which separates nucleotides according to increasing negative charge, yielded a good separation of ADP and ATP, while the AMP-containing fractions (as well as those containing ATP and ADP) were further puri-
RESULTS The introduction of 32P; into ADP and ATP of nude mouse liver or human hepatoma follows the values of their respective total absorbancies. A period of 32Pi incorporation of 2-2Y2 hr, which is sufficient to equilibrate completely the a-phosphates (E. Rapaport, unpublished data) yields a 32P-ratio of ATP/ADP (cpm ratio multiplied by %) equal to the absorbance ratio of ATP/ADP in the liver or hepatoma. The absorbance of ATP in liver or tumor, as obtained by chromatographic procedures, reflects a value of 1-3 mM based on liver or tumor wet tissue weight. The ATP value is higher when the ATP/ADP ratio is high [3-4], and drops due to hypoxia (12, 13), as is reflected in a lower ATP/ADP ratio. It is interesting to note that the ATP/ ADP ratios in the human hepatoma grown subcutaneously in the nude mice are consistently higher [3-6] than the same ratios in the corresponding livers [2-3] during similar short periods of hypoxia. This is probably a reflection of the superior ability of the tumor to undergo anaerobic glycolysis, thereby maintaining higher ATP levels upon ischemia. No difference was observed in the total ATP levels of the tumors or the corresponding livers, both giving high levels, around 3 mM. Two types of chromatographic procedures were utilized for LTPW -
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fied on a Dowex 1-X8 formate column as shown in Figs. 1 and 2. The other analytical procedure, which is demonstrated in Figs. S and 4, included initial analysis of acid-soluble nucleotides on a DEAE-cellulose column with an ammonium bicarbonate gradient elution. Fractions corresponding to ATP, ADP, and AMP were pooled, the volatile buffer was removed by repeated evaporation, and samples were subjected to PEI-cellulose thin-layer chromatography. With the latter procedure, parts of the ADP and ATP fractions were also treated with snake venom phosphodiesterase before being subjected to the PEIcellulose thin-layer chromatography. Fig. 4 includes, therefore, a direct comparison of the labeling of AMP with AMPs derived from the ADP and ATP fractions. Table 1 lists the 32P/3H values for ADP and ATP. 32P, (0.1 mCi) was introduced for 2 hr followed by the introduction of the tritiated precursor (0.5 mCi) for the time period indicated. Experiments involving nude mice carrying the hepatoma were performed with dissection of the tumor, followed immediately by excision of the liver, or, alternatively, talking the liver out first
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FIG. 2. Dowex 1-X8 (formate) column chromatography of AMP, ADP, and ATP fractions from DEAE-Sephadex column. Fractions were pooled from columns described in Fig. 1. The experiment included 50 min incorporation of [3H]adenosine (Table 1).
FIG. 4. Thin-layer chromatography of AMP, ADP, and ATP fractions from DEAE-cellulose column on PEI-cellulose plates. In addition to the AMP, ADP, and ATP spots, each fraction was digested with snake venom phosphodiesterase (SVP) followed by chromatography in the same system. Listed are 32P/3H values for the indicated region of the plate.
3124
Proc. Natt. Acad. Sci. USA 73 (1976)
'Biochemistry: Rapaport and Zamecnik
Table 1. 32P/3H ratios for ADP and ATP following in vivo radioactive labeling of nude mouse livers and hepatomas
Precursor
Adenosine
Incorporation period (min)
10 20 20 30
Hypoxanthine
Adenine
50 150 3 20 25 3 20
Table 2. ATP/ADP ratios based on their absorbancies, 32P and 3H cpm Incorporation
32P/3H Ratio Tissue
ADP
ATP
Liver Liver Liver
1.27 0.13 0.20
Tumor Liver Tumor Liver Liver Liver Liver Liver Tumor Liver Liver
0.14 0.12 0.09 0.50 1.87 0.37 1.90 0.14 0.35
1.47 0.14 0.16 0.17
0.22 0.55
Precursor Adenosine
0.04 0.08 0.53
0.22
Adenine
32p, (0.1 mCi) was injected intraperitoneally into nude mice 2 hr prior to the intravenous injection of the 3H-labeled precursors (0.5 mCi). Following the incorporation period of the tritiated precursor, livers and tumors were excised and analyzed. Values represent actual cpm ratios and were not corrected by'a factor of 2/3. Each experiment represents a different incorporation period for the tritiated precursor and was performed on a different mouse.
followed by immediate excision of the tumor. The only differences were in the ATP/ADP ratios. If the tumor is taken out first, its ATP/ADP ratio is 3-6 and the liver's is 0.5-1.5. When the liver is excised first its ATP/ADP ratio is 2-3 and the tumor's is 2-3 also. No qualitative difference, due to changes in the ATP/ADP ratios, exists as far as the 32P- and 3H-labeling patterns described in Table 1. The precursor-product- relation between ADP and ATP (and vice versa) should yield similar 32P/3H ratios following the correction by a factor of %, which accounts for the different number of phosphate groups in ADP and ATP. This is indeed the case upon labeling with [3H]adenine or [3H]hypoxanthine. [3H]Adenosine labeling, however, always yields a much smaller 32P/3H value for ATP than for ADP. In other words, there seems to be a preferential funneling of tritium label into ATP. as'compared with ADP. Another way to view this phenomenon is through the ATP/ADP ratios. The 32p cpm in the ATP fraction (multiplied by %) divided by the 32p cpm in the ADP fraction is always equal to the ratio of their total absorbancies. In the case of [3H]adenine and [3H]hypoxanthine labeled livers or tumors this ratio also equals the [3H]ATP/[3H]ADP ratio. With [3H]adenosine labeling, however, the [3H]ATP/[31i]ADP FA)/ ratios always exceed the corresponding ([32P]ATP [32P]ADP or the total absorbancy ratios. Table 2 summarizes this aspect of the data, part of which is presented in Figs. 1 and 3. It is difficult to assess an absolute quantitative value to the effect because of anoxia occurring during the 1-5 sec which elapse between severance of the tissue and its freeze clamping. During this period some ATP is converted to ADP. From livers X
and tumors which were labeled in the same mouse it seems that [3H]adenosine funneling into ATP is of a larger magnitude in the liver than in the tumor. Figs. 2 and 4 indicate that in AMP from 32P1- and [3H]adenosine-labeled tissues (livers and hepatomas) the 32P/3H ratio (multiplied by 3) is larger than the same
Tissue
A260
32P
3H
10 20 30
Liver Liver Liver Tumor Liver
2.7 4.1 0.5* 3.5 2.2 4.7 2.8 1.9 3.0 2.6 2.2
2.8 3.8 0.5* 3.2 2.1 5.1 2.6 2.1 3.5 2.5 2.0
3.7 6.0 1.7 5.9 5.3 5.6 5.6 2.1 3.1 2.6 2.0
Tumort Hypoxanthine
0.32 0.77
(min)
30
1.81 0.54 2.75
0.51
ATP/ADP Ratio
period
150 20 25 20
Liver Liver Liver Tumor Liver
Conditions were as described for Table 1. [32P]ATP/[32P]ADP represents actual cpm ratios multiplied by 2/3. * Values are unusually low because of excess bleeding caused during the removal of the tumor. t Polyoma-transformed BHK cells grown subcutaneously.
ratio for ATP. Comparison of the 32P/3H ratio in the original AMP, and in AMPs obtained from treatment of ADP and ATP with snake venom phosphodiesterase, demonstrates that the AMP and ADP carry similar32P/3H ratios, while ATP yields a much smaller 32P/3H ratio in the AMP derived from it. PQlyoma-transformed BHK cells grown subcutaneously in the nude mouse yielded similar results (Table 2) in comparison with the liver, as did the human hepatoma. DISCUSSION The experiments reported here demonstrate the existence of a mechanism by which [3H]adenosine, but not [3H]adenine or [3H]hypoxanthine, can be incorporated into ATP, in preference to its incorporation into AMP and ADP. In nude mice, from which both the liver and hepatoma were analyzed, the effect was larger in the liver. These results provide evidence for the production from adenosine of highly compartmentalized ATP, an ATP pool which is not available to adenylate kinase action. Adenylate kinase is otherwise responsible for the rapid equilibration of AMP, ADP, and ATP (14). The possibility of preferential incorporation of [3Hjadenosine into the ATP pool through an unknown pathway which provides an adenosineATP exchange has also been considered. This may be possible, but could not be a sole explanation, since 10 min incorporation of [3H]adenosine preferentially into the ATP pool was less than 2'A hr incorporation. If an unknown adenosine-ATP exchange were occurring without compartmentalization, long incorporation periods (50 min, 2Y hr) would tend to produce a smaller effect due to equilibration of the pools in the presence of adenylate kinase. The experiments reported here were performed by utilizing radioactive labeling of high specific activity, which did not cause appreciable change in the actual concentration of purine nucleotide precursors in the liver. The results therefore could not be attributed to an unknown effect of the purine nucleotide precursor per se on enzymatic pathways responsible for the rapid equilibration of AMP, ADP, and ATP. The term compartmentalizatton as used by us refers to nucleotide pools which do not mix in the cell sap by a diffusioncontrolled process. Whether the different ATP pools are in
Biochemistry: Rapaport and Zamecnik different cellular compartments separated by a membrane barrier or represent a case of kinetic compartmentalization remains to be determined. Kinetic compartmentalization was described by Atkinson (15) as resulting from association of enzymes in ways which facilitate movement of a product to the active site of the next enzyme, and hinder diffusion between the vicinity of the active site and the general cell sap. The following scheme will further clarify the interpretation of our results in terms of compartmentalization of the ATP pools. 1
Adenosine
ATP
#3
AMP, ADP 2
ATP
We have observed a restriction or blockade of the back reaction of route 1: AMP and ADP are not equilibrated with ATP derived from adenosine. Route 2 represents the normal rapid equilibrium obtained through the action of adenylate kinase. Therefore, since route 1 is clearly shown to exist, route 3 must be blocked, meaning separate ATP pools. If route 3 represented an equilibrium situation, route 1 could not have been detected. Furthermore, the results presented here imply that at least some of the adenosine kinase is compartmentalized, since AMP formed from adenine and phosphoribosyl pyrophosphate in the presence of adenine phosphoribosyltransferase does not produce compartmentalized ATP, while AMP formed from adenosine and ATP in the presence of adenosine kinase yields compartmentalized ATP. The first step in the metabolism of adenosine, the formation of AMP by adenosine kinase action (16), has not been reported to be subject to feedback regulation. Adenine and hypoxanthine, on the other hand, yield AMP and IMP in the presence of adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase, respectively. These reactions are sensitive to the availability of phosphoribosyl pyrophosphate, as is the de novo synthesis of purines. Phosphoribosyl pyrophosphate in turn is produced from ATP and ribose 5phosphate in the presence of phosphoribosyl pyrophosphate synthetase, an enzyme known to be regulated by the relative concentration of AMP, ADP, and ATP (17, 18). These differences, which would enable the metabolism of adenosine and not that of hypoxanthine or adenine to be dependent on substrate concentration, are presumably responsible to the reported remarkable increase in cellular ATP upon treatment with 0.5-1.0 mM adenosine (1-5). Quantitatively, an increase in hepatic ATP levels from 2.1 to 6.5 mM, as well as a 3-fold increase in the ATP/ADP ratio, has been reported (3). Our results, however, indicate that the majority of the increase in ATP produced from adenosine is compartmentalized and thus may not be available for certain metabolic functions. The experimental results reported by Lund et al. (3) are also in agreement with the concept of compartmentalization. The 3-fold increase in the ATP/ADP ratios would exist only if part of the ATP responsible for the dramatic increase in total ATP levels is not subject to the metabolic processes (e.g., adenylate kinase, hexokinase, phosphofructokinase) which yield normal ATP/ADP ratios of 2-3. The existence of independent ATP pools, a small nuclear pool and a large cytoplasmic pool, has been disputed in the past. Plagemann (1) has presented evidence for the existence of separate nucleotide pools in Novikoff rat hepatoma cells in
Proc. Nati. Acad. Sci. USA 73 (1976)
3125
culture while Ove et al. (19) have indicated that nucleotides in the nucleus and cytoplasm are in rapid equilibrium in rat liver. Utilizing different. experimental techniques, we have demonstrated that the incorporation of adenosine into acidsoluble nucleotides in nude mouse liver and a fast growing human hepatoma reveals a high degree of compartmentalization of the ATP pools, while AMP and ADP pools are com-
pletely equilibrated.
We have found that the levels of ATP in the nude mouse liver and human hepatoma grown subcutaneously are very similar. From the absorbancies of ATP per gram of wet tissue weight these values were shown to be 2-S mM under conditions where the ATP/ADP ratios were about 3. Our observation that the total ATP level of a tissue arrested in Go phase of the cell cycle (mouse liver) is similar to its level in a fast-growing hepatoma may provide added importance to our finding that there is a difference in the level of ATP compartmentalization in the two tissues. Adenosine-induced ATP increase is substantially higher in liver than in the tumor; most of the studies were performed on a human hepatoma. However, polyoma-transformed BHK cells grown subcutaneously in the nude mouse yielded similar results. Depletion of some compartmentalized ATP pools in the tumor relative to the nongrowing liver may suggest that cell division or some processes leading to it utilize compartmentalized ATP pools. We gratefully acknowledge the expert technical assistance of Ms. Sandra K. Svihovec. We are indebted to Dr. Nancy L. R. Bucher for her help in carrying out some of the biological experiments and for helpful discussions. We are grateful to Dr. Jesse F. Scott for providing some of the research facilities, and for critical review of the manuscript. This work was supported by National Cancer Institute Virus Contract Program (no. N01 CP-33-66) and the United States Energy Research and Development Administration contract [no. E(11-1) 2404]. This is Publication no. 1504 of the Cancer Commission of Harvard University. 1. Plagemann, P. G. W. (1972) J. Cell Biol. 52, 131-146. 2. Chagoya de Sanchez, V., Brunner, A. & Pina, E. (1972) Biochem. Blophys. Res. Commun. 46,1441-1445. 3. Lund, P., Cornell, N. W. & Krebs, H. A. (1975) Biochem. J. 152, 593-599.
4. Wilkening, J., Nowack, J. & Decker, K. (1975) Biochim. Blophys. Acta 392, 299-309. 5. Grummt, I. & Grummt, F. (1976) Cell 7,447-453. 6. Ishii, K. & Green, H. (1973) J. Cell Sci. 13, 429-439. 7. Brooks, R. F. (1975) J. Cell. Physiol. 86, 369-377. 8. Chan, T. S., Ishit, K., Long, C. & Green, H. (1973) J. Cell. Physiol.
81,315-321.
9. Murray, A. W., Elliott, D. C. & Atkinson, M. R. (1970) Prog. Nucleic Acid Res. Mol. Biol. 10, 87-119. 10. Bucher, N. L. R. & Swaffield M. N. (1966) Biochim. Blophys. Acta 129, 445-459. 11. Wollenberger, A., Ristau, 0. & Schoffa, G. (1960) Pfluegers Arch. Ges. Physiol. Menschen Tiere 270,399-412. 12. Fox, I. H. (1974) Nutr. Metabol. 16,65-78. 13. Weber, G., Stubbs, M. & Morris, H. P. (1971) Cancer Res. 31, 2177-2183. 14. Atkinson, D. E. (1966) Annu. Rev. Biochem. 35,85-124. 15. Atkinson, D. E. (1968) Biochemistry 7,4030-4034. 16. Caputto R. (1951) J. Biol. Chem. 189,801-814. 17. Atkinson, D. E. & Fall, L. (1967) J. Biol. Chem. 242, 32413244. 18. Fox, I. H. & Kelley, W. N. (1972) J. Biol. Chem. 247, 21262131. 19. Ove, P., Takai, S., Umeda, T. & Lieberman, I. (1967) J. Biol. Chem. 242, 4963-4971.
Incorporation of adenosine into ATP: Formation of compartmentalized ATP (radioactive labeling in vivo/adenine nucleotides/tumors)
ELIEZER RAPAPORT AND PAUL C. ZAMECNIK The John Collins Warren Laboratories of the Huntington Memorial Hospital of Harvard University at the Massachusetts General Hospital, Boston, Mass. 02114
Contributed by Paul C. Zamecnik, June 28, 1976
ABSTRACT The incorporation of [3H]adenosine, [3H]adenine, and [3Hlhypoxanthine into adenine nucleotides of nude (athymic) mouse liver and human hepatoma grown subcutaneously in nude mice was studied. 3H and 32P radioactive labeling in vivo of acid-soluble nucleotides followed by chromaprocedures indicated that, in contrast to [3Hjadenine tographic and [ H]hypoxanthine, [3H]adenosine is preferentially incorporated into ATP in comparison with its incorporation into AMP and ADP. This phenomenon, as well as complementing the recently reported 3fold increase in total cellular ATP upon treatment with 0.5-1.0 mM concentrations of adenosine, indicates the formation from adenosine of compartmentalized ATP that is not produced from either adenine or hypoxanthine. The observed effect is of larger magnitude in the growth-arrested normal liver than in the actively growing tumor.
MATERIALS AND METHODS Nude (athymnic) mice were obtained from Dr. WendallFarrow of Life Sciences Research Laboratories, through the auspices of the Office of Program Resources of the Virus Cancer Program, National Cancer Institute. A human malignant hepatoma cell line was developed from a primary tumor by Dr. Kurt Isselbacher's laboratory. The cell line is malignant, as judged by progressive infiltration and lethality in the nude mice, and is capable of synthesizing certain proteins characteristic of human liver. About 5 X 107 cells were injected subcutaneously into nude mice (16-19 g). After 10-14 days a solid tumor of 0.5-1.0 g developed and mice were used at this stage. Radioactive chemicals were supplied by New England Nuclear (Boston, Mass.). 32P1 (0.1 mCi in 0.9% NaCl) was injected intraperitoneally, and 3H-labeled precursors (0.5 mCi in 0.9% NaCl) were injected intravenously in a total volume of 0.2 ml. All 3H-labeled compounds were of the highest specific activity available (15-25 Ci/mmol). These procedures as well as the final dissection and freeze-clamping of liver or tumor were carried out under ether anesthesia in an atmosphere of 100% oxygen to minimize hypoxia (10). Livers and tumors were excised and within 1-5 sec were freeze-clamped between heavy aluminum plates precooled in liquid nitrogen (11). The frozen tissue was pulverized in a mortar at solid CO2 temperature and extracted for at least 30 min with ice-cold 0.5 M perchloric acid. Following removal of cell debris by centrifugation, the solution was neutralized with 7 M KOH. Two procedures were used for analyzing acid-soluble nucleotides, and both gave similar results. Acid-soluble nucleotides were analyzed on a DEAESephadex A-25 column, eluted with a linear gradient of 0.1-0.6 M NaCl in 0.05 M Tris-HCl, 0.002 M EDTA, and 7 M urea, pH 7.6. This procedure affords a separation according to increasing negative charge. The fractions containing ADP and ATP were well separated. The fractions containing AMP (as well as those containing ADP and ATP) were diluted 5-fold and applied on top of a Dowex 1-X8 (formate) column. Elution with a linear gradient of 0.0-2.0 M ammonium formate, pH 5.0, afforded further purification of these nucleotides. The second sequence which was utilized more often included initial chromatography of acid-soluble nucleotides on a DEAE-cellulose column eluted with a linear gradient of 0.0-0.5 M ammonium bicarbonate, pH 8.1. Appropriate fractions were pooled and the buffer was removed by repeated evaporation and lyophilization. The dry residues were dissolved in a small volume of water and part of the solution was subjected to snake venom phosphodiesterase digestion followed by thin-layer chromatography on PEI-cellulose. Elution was performed with water followed by 1 M LiCl. The plate was cut and the small pieces were eluted with 4 M ammonium hydroxide for at least 1 hr, before measuring the
Several recent reports have indicated that treatment of different tissues with 0.5-1.0 mM concentrations of adenosine caused a marked increase in their ATP levels (1-5). It was shown that this increase, which was also reflected in high ATP/ADP ratios, could not be attained with other adenine nucleotide precursors, namely, adenine, hypoxanthine, or inosine (3). Adenosine has been found to be lethal to cells in culture at a low concentration (6), as the result of a metabolic imbalance which shuts off pyrimidine biosynthesis. This lethality was abolished in mutants which did not contain adenosine kinase and was thus attributed to a phosphorylated intermediate (6). The reported stimulatory effect of small adenosine concentrations on DNA synthesis in the presence of serum (7) can be attributed at least in part to inosine formed from adenosine with adenosine deaminase present in serum in tissue cultures (Rapaport and Zamecnik,
unpublished data). Adenosine kinase and adenosine deaminase
are the enzymes known to metabolize adenosine in mammalian tissue (8), yielding AMP and inosine, respectively. Inosine is an established intermediate in the purine salvage pathway (9). We have utilized high specific activity radioactive precursors to study the incorporation of adenosine into ATP in nude mouse
liver and subcutaneous, fast-growing human hepatoma in the nude mouse. The results indicate that in both tissues [3H] adenosine but not [3H]adenine or [3H]hypoxanthine is preferentially incorporated into ATP relative to its incorporation into AMP or ADP. Thus, the existence of a pathway which leads to a compartmentalized ATP pool from adenosine is indicated. This phenomenon is of a larger magnitude in the liver than in the hepatoma grown in the same nude mouse. Polyomatransformed baby hamster kidney cells (BHK21/C13) grown in the nude mouse yielded similar results. The direct comparison of the total ATP levels and the metabolically significant ATP/ADP ratios in liver and tumor of the same live animal, as well as the different magnitudes of ATP
compartmentalization in the liver and tumor, have been studied.
3122
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Biochemistry: Rapaport and Zamecnik ATP
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60
3123
to
20
30
50
40
60
70
90
60
100
FRACTION NUMBER (5 ml each)
FIG. 1. DEAE-Sephadex-7 M urea column chromatography of acid-soluble nucleotides from nude mouse liver (a) and tumor (b). Radioactive labeling was performed with 0.1 mCi of 32Pj for 2 hr followed by 0.5 mCi of [3H]adenosine for 30 min. The tumor was excised first; hypoxia caused by some bleeding is the probable reason for the low ATP/ADP ratio in the liver.
FIG. 3. DEAE-cellulose column chromatography of acid-soluble nucleotides from nude mouse liver (a) and tumor (b). Radioactive labeling was performed as described in the legend to Fig. 1 except that [3H]adenosine was injected for a period of 20 min before excision of the liver, followed by removal of the tumor.
radioactivity of the ammonium hydroxide eluate in Bray's scintillation fluid. Snake venom phosphodiesterase digestions were performed in 0.05 M Tris-HCl, 0.05 M MgCl2, pH 8.4, at 300 for 1 hr.
analyzing the acid-soluble nucleotides. DEAE-Sephadex-7 M urea column chromatography, which separates nucleotides according to increasing negative charge, yielded a good separation of ADP and ATP, while the AMP-containing fractions (as well as those containing ATP and ADP) were further puri-
RESULTS The introduction of 32P; into ADP and ATP of nude mouse liver or human hepatoma follows the values of their respective total absorbancies. A period of 32Pi incorporation of 2-2Y2 hr, which is sufficient to equilibrate completely the a-phosphates (E. Rapaport, unpublished data) yields a 32P-ratio of ATP/ADP (cpm ratio multiplied by %) equal to the absorbance ratio of ATP/ADP in the liver or hepatoma. The absorbance of ATP in liver or tumor, as obtained by chromatographic procedures, reflects a value of 1-3 mM based on liver or tumor wet tissue weight. The ATP value is higher when the ATP/ADP ratio is high [3-4], and drops due to hypoxia (12, 13), as is reflected in a lower ATP/ADP ratio. It is interesting to note that the ATP/ ADP ratios in the human hepatoma grown subcutaneously in the nude mice are consistently higher [3-6] than the same ratios in the corresponding livers [2-3] during similar short periods of hypoxia. This is probably a reflection of the superior ability of the tumor to undergo anaerobic glycolysis, thereby maintaining higher ATP levels upon ischemia. No difference was observed in the total ATP levels of the tumors or the corresponding livers, both giving high levels, around 3 mM. Two types of chromatographic procedures were utilized for LTPW -
f
150
I 10
Pi MARKERS
AMP
ADP
C)
C0
AMP
4~~~~~00
I I
ItI.."
LOP
150
fied on a Dowex 1-X8 formate column as shown in Figs. 1 and 2. The other analytical procedure, which is demonstrated in Figs. S and 4, included initial analysis of acid-soluble nucleotides on a DEAE-cellulose column with an ammonium bicarbonate gradient elution. Fractions corresponding to ATP, ADP, and AMP were pooled, the volatile buffer was removed by repeated evaporation, and samples were subjected to PEI-cellulose thin-layer chromatography. With the latter procedure, parts of the ADP and ATP fractions were also treated with snake venom phosphodiesterase before being subjected to the PEIcellulose thin-layer chromatography. Fig. 4 includes, therefore, a direct comparison of the labeling of AMP with AMPs derived from the ADP and ATP fractions. Table 1 lists the 32P/3H values for ADP and ATP. 32P, (0.1 mCi) was introduced for 2 hr followed by the introduction of the tritiated precursor (0.5 mCi) for the time period indicated. Experiments involving nude mice carrying the hepatoma were performed with dissection of the tumor, followed immediately by excision of the liver, or, alternatively, talking the liver out first
4 20
2 0 83
ADP
SVP TREATED
0
ADP AMP
ATP
C>
0 "2
0
0
0
SVP TREATED
ATP
CD C 44 ,D48
5
40
45
20
DISTANCE FROM OR/GIN (cm)
FRACTIONs NUJMBERf ( 5 m/ eoch)f
FIG. 2. Dowex 1-X8 (formate) column chromatography of AMP, ADP, and ATP fractions from DEAE-Sephadex column. Fractions were pooled from columns described in Fig. 1. The experiment included 50 min incorporation of [3H]adenosine (Table 1).
FIG. 4. Thin-layer chromatography of AMP, ADP, and ATP fractions from DEAE-cellulose column on PEI-cellulose plates. In addition to the AMP, ADP, and ATP spots, each fraction was digested with snake venom phosphodiesterase (SVP) followed by chromatography in the same system. Listed are 32P/3H values for the indicated region of the plate.
3124
Proc. Natt. Acad. Sci. USA 73 (1976)
'Biochemistry: Rapaport and Zamecnik
Table 1. 32P/3H ratios for ADP and ATP following in vivo radioactive labeling of nude mouse livers and hepatomas
Precursor
Adenosine
Incorporation period (min)
10 20 20 30
Hypoxanthine
Adenine
50 150 3 20 25 3 20
Table 2. ATP/ADP ratios based on their absorbancies, 32P and 3H cpm Incorporation
32P/3H Ratio Tissue
ADP
ATP
Liver Liver Liver
1.27 0.13 0.20
Tumor Liver Tumor Liver Liver Liver Liver Liver Tumor Liver Liver
0.14 0.12 0.09 0.50 1.87 0.37 1.90 0.14 0.35
1.47 0.14 0.16 0.17
0.22 0.55
Precursor Adenosine
0.04 0.08 0.53
0.22
Adenine
32p, (0.1 mCi) was injected intraperitoneally into nude mice 2 hr prior to the intravenous injection of the 3H-labeled precursors (0.5 mCi). Following the incorporation period of the tritiated precursor, livers and tumors were excised and analyzed. Values represent actual cpm ratios and were not corrected by'a factor of 2/3. Each experiment represents a different incorporation period for the tritiated precursor and was performed on a different mouse.
followed by immediate excision of the tumor. The only differences were in the ATP/ADP ratios. If the tumor is taken out first, its ATP/ADP ratio is 3-6 and the liver's is 0.5-1.5. When the liver is excised first its ATP/ADP ratio is 2-3 and the tumor's is 2-3 also. No qualitative difference, due to changes in the ATP/ADP ratios, exists as far as the 32P- and 3H-labeling patterns described in Table 1. The precursor-product- relation between ADP and ATP (and vice versa) should yield similar 32P/3H ratios following the correction by a factor of %, which accounts for the different number of phosphate groups in ADP and ATP. This is indeed the case upon labeling with [3H]adenine or [3H]hypoxanthine. [3H]Adenosine labeling, however, always yields a much smaller 32P/3H value for ATP than for ADP. In other words, there seems to be a preferential funneling of tritium label into ATP. as'compared with ADP. Another way to view this phenomenon is through the ATP/ADP ratios. The 32p cpm in the ATP fraction (multiplied by %) divided by the 32p cpm in the ADP fraction is always equal to the ratio of their total absorbancies. In the case of [3H]adenine and [3H]hypoxanthine labeled livers or tumors this ratio also equals the [3H]ATP/[3H]ADP ratio. With [3H]adenosine labeling, however, the [3H]ATP/[31i]ADP FA)/ ratios always exceed the corresponding ([32P]ATP [32P]ADP or the total absorbancy ratios. Table 2 summarizes this aspect of the data, part of which is presented in Figs. 1 and 3. It is difficult to assess an absolute quantitative value to the effect because of anoxia occurring during the 1-5 sec which elapse between severance of the tissue and its freeze clamping. During this period some ATP is converted to ADP. From livers X
and tumors which were labeled in the same mouse it seems that [3H]adenosine funneling into ATP is of a larger magnitude in the liver than in the tumor. Figs. 2 and 4 indicate that in AMP from 32P1- and [3H]adenosine-labeled tissues (livers and hepatomas) the 32P/3H ratio (multiplied by 3) is larger than the same
Tissue
A260
32P
3H
10 20 30
Liver Liver Liver Tumor Liver
2.7 4.1 0.5* 3.5 2.2 4.7 2.8 1.9 3.0 2.6 2.2
2.8 3.8 0.5* 3.2 2.1 5.1 2.6 2.1 3.5 2.5 2.0
3.7 6.0 1.7 5.9 5.3 5.6 5.6 2.1 3.1 2.6 2.0
Tumort Hypoxanthine
0.32 0.77
(min)
30
1.81 0.54 2.75
0.51
ATP/ADP Ratio
period
150 20 25 20
Liver Liver Liver Tumor Liver
Conditions were as described for Table 1. [32P]ATP/[32P]ADP represents actual cpm ratios multiplied by 2/3. * Values are unusually low because of excess bleeding caused during the removal of the tumor. t Polyoma-transformed BHK cells grown subcutaneously.
ratio for ATP. Comparison of the 32P/3H ratio in the original AMP, and in AMPs obtained from treatment of ADP and ATP with snake venom phosphodiesterase, demonstrates that the AMP and ADP carry similar32P/3H ratios, while ATP yields a much smaller 32P/3H ratio in the AMP derived from it. PQlyoma-transformed BHK cells grown subcutaneously in the nude mouse yielded similar results (Table 2) in comparison with the liver, as did the human hepatoma. DISCUSSION The experiments reported here demonstrate the existence of a mechanism by which [3H]adenosine, but not [3H]adenine or [3H]hypoxanthine, can be incorporated into ATP, in preference to its incorporation into AMP and ADP. In nude mice, from which both the liver and hepatoma were analyzed, the effect was larger in the liver. These results provide evidence for the production from adenosine of highly compartmentalized ATP, an ATP pool which is not available to adenylate kinase action. Adenylate kinase is otherwise responsible for the rapid equilibration of AMP, ADP, and ATP (14). The possibility of preferential incorporation of [3Hjadenosine into the ATP pool through an unknown pathway which provides an adenosineATP exchange has also been considered. This may be possible, but could not be a sole explanation, since 10 min incorporation of [3H]adenosine preferentially into the ATP pool was less than 2'A hr incorporation. If an unknown adenosine-ATP exchange were occurring without compartmentalization, long incorporation periods (50 min, 2Y hr) would tend to produce a smaller effect due to equilibration of the pools in the presence of adenylate kinase. The experiments reported here were performed by utilizing radioactive labeling of high specific activity, which did not cause appreciable change in the actual concentration of purine nucleotide precursors in the liver. The results therefore could not be attributed to an unknown effect of the purine nucleotide precursor per se on enzymatic pathways responsible for the rapid equilibration of AMP, ADP, and ATP. The term compartmentalizatton as used by us refers to nucleotide pools which do not mix in the cell sap by a diffusioncontrolled process. Whether the different ATP pools are in
Biochemistry: Rapaport and Zamecnik different cellular compartments separated by a membrane barrier or represent a case of kinetic compartmentalization remains to be determined. Kinetic compartmentalization was described by Atkinson (15) as resulting from association of enzymes in ways which facilitate movement of a product to the active site of the next enzyme, and hinder diffusion between the vicinity of the active site and the general cell sap. The following scheme will further clarify the interpretation of our results in terms of compartmentalization of the ATP pools. 1
Adenosine
ATP
#3
AMP, ADP 2
ATP
We have observed a restriction or blockade of the back reaction of route 1: AMP and ADP are not equilibrated with ATP derived from adenosine. Route 2 represents the normal rapid equilibrium obtained through the action of adenylate kinase. Therefore, since route 1 is clearly shown to exist, route 3 must be blocked, meaning separate ATP pools. If route 3 represented an equilibrium situation, route 1 could not have been detected. Furthermore, the results presented here imply that at least some of the adenosine kinase is compartmentalized, since AMP formed from adenine and phosphoribosyl pyrophosphate in the presence of adenine phosphoribosyltransferase does not produce compartmentalized ATP, while AMP formed from adenosine and ATP in the presence of adenosine kinase yields compartmentalized ATP. The first step in the metabolism of adenosine, the formation of AMP by adenosine kinase action (16), has not been reported to be subject to feedback regulation. Adenine and hypoxanthine, on the other hand, yield AMP and IMP in the presence of adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase, respectively. These reactions are sensitive to the availability of phosphoribosyl pyrophosphate, as is the de novo synthesis of purines. Phosphoribosyl pyrophosphate in turn is produced from ATP and ribose 5phosphate in the presence of phosphoribosyl pyrophosphate synthetase, an enzyme known to be regulated by the relative concentration of AMP, ADP, and ATP (17, 18). These differences, which would enable the metabolism of adenosine and not that of hypoxanthine or adenine to be dependent on substrate concentration, are presumably responsible to the reported remarkable increase in cellular ATP upon treatment with 0.5-1.0 mM adenosine (1-5). Quantitatively, an increase in hepatic ATP levels from 2.1 to 6.5 mM, as well as a 3-fold increase in the ATP/ADP ratio, has been reported (3). Our results, however, indicate that the majority of the increase in ATP produced from adenosine is compartmentalized and thus may not be available for certain metabolic functions. The experimental results reported by Lund et al. (3) are also in agreement with the concept of compartmentalization. The 3-fold increase in the ATP/ADP ratios would exist only if part of the ATP responsible for the dramatic increase in total ATP levels is not subject to the metabolic processes (e.g., adenylate kinase, hexokinase, phosphofructokinase) which yield normal ATP/ADP ratios of 2-3. The existence of independent ATP pools, a small nuclear pool and a large cytoplasmic pool, has been disputed in the past. Plagemann (1) has presented evidence for the existence of separate nucleotide pools in Novikoff rat hepatoma cells in
Proc. Nati. Acad. Sci. USA 73 (1976)
3125
culture while Ove et al. (19) have indicated that nucleotides in the nucleus and cytoplasm are in rapid equilibrium in rat liver. Utilizing different. experimental techniques, we have demonstrated that the incorporation of adenosine into acidsoluble nucleotides in nude mouse liver and a fast growing human hepatoma reveals a high degree of compartmentalization of the ATP pools, while AMP and ADP pools are com-
pletely equilibrated.
We have found that the levels of ATP in the nude mouse liver and human hepatoma grown subcutaneously are very similar. From the absorbancies of ATP per gram of wet tissue weight these values were shown to be 2-S mM under conditions where the ATP/ADP ratios were about 3. Our observation that the total ATP level of a tissue arrested in Go phase of the cell cycle (mouse liver) is similar to its level in a fast-growing hepatoma may provide added importance to our finding that there is a difference in the level of ATP compartmentalization in the two tissues. Adenosine-induced ATP increase is substantially higher in liver than in the tumor; most of the studies were performed on a human hepatoma. However, polyoma-transformed BHK cells grown subcutaneously in the nude mouse yielded similar results. Depletion of some compartmentalized ATP pools in the tumor relative to the nongrowing liver may suggest that cell division or some processes leading to it utilize compartmentalized ATP pools. We gratefully acknowledge the expert technical assistance of Ms. Sandra K. Svihovec. We are indebted to Dr. Nancy L. R. Bucher for her help in carrying out some of the biological experiments and for helpful discussions. We are grateful to Dr. Jesse F. Scott for providing some of the research facilities, and for critical review of the manuscript. This work was supported by National Cancer Institute Virus Contract Program (no. N01 CP-33-66) and the United States Energy Research and Development Administration contract [no. E(11-1) 2404]. This is Publication no. 1504 of the Cancer Commission of Harvard University. 1. Plagemann, P. G. W. (1972) J. Cell Biol. 52, 131-146. 2. Chagoya de Sanchez, V., Brunner, A. & Pina, E. (1972) Biochem. Blophys. Res. Commun. 46,1441-1445. 3. Lund, P., Cornell, N. W. & Krebs, H. A. (1975) Biochem. J. 152, 593-599.
4. Wilkening, J., Nowack, J. & Decker, K. (1975) Biochim. Blophys. Acta 392, 299-309. 5. Grummt, I. & Grummt, F. (1976) Cell 7,447-453. 6. Ishii, K. & Green, H. (1973) J. Cell Sci. 13, 429-439. 7. Brooks, R. F. (1975) J. Cell. Physiol. 86, 369-377. 8. Chan, T. S., Ishit, K., Long, C. & Green, H. (1973) J. Cell. Physiol.
81,315-321.
9. Murray, A. W., Elliott, D. C. & Atkinson, M. R. (1970) Prog. Nucleic Acid Res. Mol. Biol. 10, 87-119. 10. Bucher, N. L. R. & Swaffield M. N. (1966) Biochim. Blophys. Acta 129, 445-459. 11. Wollenberger, A., Ristau, 0. & Schoffa, G. (1960) Pfluegers Arch. Ges. Physiol. Menschen Tiere 270,399-412. 12. Fox, I. H. (1974) Nutr. Metabol. 16,65-78. 13. Weber, G., Stubbs, M. & Morris, H. P. (1971) Cancer Res. 31, 2177-2183. 14. Atkinson, D. E. (1966) Annu. Rev. Biochem. 35,85-124. 15. Atkinson, D. E. (1968) Biochemistry 7,4030-4034. 16. Caputto R. (1951) J. Biol. Chem. 189,801-814. 17. Atkinson, D. E. & Fall, L. (1967) J. Biol. Chem. 242, 32413244. 18. Fox, I. H. & Kelley, W. N. (1972) J. Biol. Chem. 247, 21262131. 19. Ove, P., Takai, S., Umeda, T. & Lieberman, I. (1967) J. Biol. Chem. 242, 4963-4971.
