Proc. Nati. Acad. Sci. USA Vol. 73, No. 11, pp. 3984-3988, November 1976 Biochemistry

Presence of diadenosine 5',5"'-Pl,P4-tetraphosphate (Ap4A) in mammalian cells in levels varying widely with proliferative activity of the tissue: A possible positive "pleiotypic activator" (signal nucleotide/growth regulation/adenine nucleotides) 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, September 10, 1976

ABSTRACT An accurate assay of diadenosine 5',5"- tuting the well-known pyrophosphate exchange (Eq. 1). P',P4-tetraphosphate [A(5') pppp(5')AJ, which was shown to be formed in vitro in the backreaction of the amino acid activation pppA + aa, + Eaal :± aal-pA-Eaa1 + pp [1] step, has been developed in various cell lines in culture and in + pppA normal mouse liver or hepatoma in vivo. Use of radioactive aal-pA-Eaal labeling of acid-soluble nucleotides to high specific activity ~ 'A(5')pppp(5')A + aa, + Eaal [2] followed by chromatographic separation techniques yielded levels of Ap4A varying from 5 to 0.05 1M (from 30 pmol/mg of However, in in vitro amino acid activation systems ATP was rotein to 0.15 pmol), depending on the doubling time of the cell shown to participate in the backreaction as well (Eq. 2), yielding line or the proliferative state of the cells. The levels of Ap4A in Ap4A, which was identified as a byproduct of the first step in cells is inversely related to their doubling time, varying from protein synthesis in vitro and in vivo (Escherichia coli and rat 0.1 X 10-4 of the cellular ATP levels in slowly growing cells to liver slice) (11-15). A wide variety of nucleoside 5'-diphosphates 20 X 10-4 of the ATP levels of cells with rapid doubling times. and 5'-triphosphates were found to compete successfully with The steady-state levels of ATP of different cell lines, although showing some fluctuations, are not related to the doubling time pyrophosphate in the backreaction of the amino acid activation of the cells. Arrest of cellular proliferation by serum deprivation step (14-16). Furthermore, pure Ap4A is capable of substituting or amino acid starvation, which does not alter the cellular ATP for ATP in the formation of aminoacyl adenylates (backreaction levels more than 2-fold, does nevertheless cause a decrease of of Eq. 2), thus supporting aminoacylation (S. M. Hecht, personal 30 to 50-fold in the Ap4A levels. Inhibition of protein synthesis communication). by pactamycin or puromycin, or inhibition of DNA synthesis It is interesting to note that the encysted embryos of the brine by hydroxyurea, leads to a more dramatic decrease of 50 to 100-fold in intracellular Ap4A levels. The metabolic lability of shrimp Artemia salina contain large amounts of diguanosine Ap4A is also demonstrated by its rapid depletion after decreases 5',5"'-P1,P4-tetraphosphate (Gp4G), which is their major in the ATP/ADP ratio. The possibility of Ap4A being a meta- acid-soluble nucleotide (17). Gp4G is synthesized by a specific bolic "signal nucleotide" that is formed at the onset of protein enzyme present in the yolk platelets during oogenesis and is synthesis and is active in positive growth regulation (positive utilized in the purine nucleotide buildup during development pleiotypic activation) is discussed. (18). Specific enzymes, capable of hydrolyzing Ap4A as well as Gp4G to ATP and AMP or GTP and GMP, respectively, have Several acid-soluble nucleotides act as specific signals in the recently been isolated from rat liver and Artemia salina regulation of specific sets of metabolic reactions (1). Since the (19). discovery of 3':5'-cyclic AMP (see ref. 2), numerous reports have We have used high specific activity radioactive labeling of followed on the effect of various 3':5'-cyclic nucleoside mo- acid-soluble nucleotides followed by chromatographic proce- nophosphates on the regulation of different biological functions, dures to assay the level of Ap4A in a variety of cells. The level including cellular proliferation (3, 4). The stringent response of Ap4A was in general found to be inversely related to the in bacteria, whereby a set of intracellular biochemical events doubling times of the cells. Treatment that led to the arrest of is controlled by the availability of an essential amino acid, leads cellular growth resulted in a large drop in intracellular Ap4A to the accumulation of ppGpp upon amino acid starvation in without significantly affecting the steady-state levels of cellular stringent but not in relaxed strains of bacteria (5). This nucle- ATP. otide, however, has not been identified in various mammalian cells in vivo (6, 7). Nevertheless, various mammalian cells in MATERIALS AND METHODS culture respond to environmental conditions that tend to slow growth in a fashion similar to the bacterial stringent response, Diadenosine 5',5w-P1,P4-tetraphosphate (Ap4A) was prepared in what has been termed a "pleiotypic response" (8). The fol- by a modification of the reported procedure (20). Disodium lowing report demonstrates the existence in mammalian cells ATP was converted to the tributylammonium salt and reacted of a highly phosphorylated nucleotide, related to ATP and with an equivalent of the 4-morpholine-N,N'-dicyclohexyl- aminoacyl adenylates, which may play a role in "positive carboxamidine salt of adenosine 5'-phosphoromorpholidate in pleiotypic activation" of cellular proliferation. dry pyridine for 3 days at room temperature. The first step in protein synthesis includes the formation of Cell Culture. A variety of monolayer cell lines were grown the aminoacyl adenylate (9, 10), with the backreaction consti- in 75cm2 Falcon flasks in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, without antibiotics. Abbreviations: Ap4A, diadenosine 5',5"'-Pl,P4-tetraphosphate; BHK, Chinese hamster ovary cells were grown in F-10 medium baby hamster kidney 21/13 cell line; Py-BHK, polyoma-transformed supplemented with 10% calf serum. In those experiments in BHK 21/13. which the incorporation in vivo of [3H1thymidine or [3H]leucine 3984 Downloaded by guest on September 26, 2021 Biochemistry: Rapaport and Zamecnik Proc. Natl. Acad. Sci. USA 73 (1976) 3985

ATP 8 ATP AP4A 1 2000 A 1 2000 1500 500 1000 i000 .4.4 '900 900 800 0-w 55--63 700 1% - 700 -600 11I? 500 v _- -%f 500 _. _. 400 '? -400 " I! He B 300 S 300 - ADP I IGTP atI C 200 k 200 ;100a | Hii. '6°&100 4 .9 k 050.5 r~~~~~~~~~~~~6 O.I 50 05 50 .40 04J 40 ,q 0.3 - I 30 N 0.3 -30 0B2eAP; 4A 20 % 02 20 z 0.1 10 0.1 1- 2)10 I 0 1040 50 60 70 80 90 100 0 100 CFAMr NUMPER (5,/ each) PRACTOV AVAAWR(f5v ech) FIG. 2. DEAE-cellulose column chromatography of fractions FIG. 1. DEAE-Sephadex-7 M urea column chromatography of 55-63 from the DEAE-Sephadex-7 M urea column described in Fig. acid-soluble nucleotides from Py-BHK cells. Unlabeled carriers of 1. ADP, ATP, GTP, and Ap4A were added at the beginning of the ex- traction procedure. The cells were treated with 0.3 mCi of phosphodiesterase digestion was performed in 0.05 M Tris-HCl, [2-:lHladenine and 0.5 mCi of 32p; for 6 hr. 0.05 M MgCl2, pH 8.4, at 300 for 1 hr. Cells grown on glass cover slips incorporated [3H]thymidine was determined, the cells were grown in 55-cm2 petri dishes (5 ,gCi) for 1 hr and [3H]leucine (10 uCi) for 40 min. The cover containing sterile 5-cm2 glass cover slips. The cover slips were slips were washed with balanced salt solution and the cells were removed before radioactive labeling of acid-soluble nucleotides, lysed with 1.5 ml of 1.0% sodium dodecyl sulfate in 0.1 M so- incubated in the appropriate medium, and labeled with either dium acetate (pH 5.0). The solution was then added to 7.5 ml [3H]thymidine or [3H]leucine. of 10% trichloroacetic acid, filtered through a glass fiber filter, Acid-soluble nucleotides in mouse liver or in human hepa- and washed with 5% trichloroacetic acid; radioactivity was then toma (C3A) grown subcutaneously in the nude mouse were determined. radioactively labeled as described (21). Analytical Procedures. Individual cultures were labeled with RESULTS 0.3 mCi of [2-3H]adenine (27 Ci/mmol) and 32p; (0.4 mCi, Ap4A Is Present in a Variety of Mammalian Cells. Acid- carrier free) for 6 hr. The medium was carefully removed, soluble nucleotides were labeled with high specific activity leaving a thin film of fluid behind at each washing. Two [3H]adenine and 32Pi and then were chromatographically an- washings of approximately 15 ml each of warm medium were alyzed. The use of [3H]adenine yielded higher levels of ra- done in a 370 room. Lack of care and speed in this step resulted dioactive labeling of ATP and Ap4A then was achieved by the in a lowering of the consistently high (>10) ATP/ADP ratios. use of comparable amounts of either [3H]adenosine or [3H] The last washing was immediately followed by the addition of hypoxanthine (21). The mixture of the radioactively labeled 10 ml of ice-cold 0.5 M perchloric acid. Acid-soluble nucleotides nucleotides was fractionated on a DEAE-Sephadex-7 M urea were extracted for 1 hr at 4°, the perchlorate was neutralized column, which yielded a separation of the ADP, ATP, and GTP with 7 M KOH, and the potassium perchlorate was removed fractions (Fig. 1). Due to their similar negative charge (-4), by centrifugation. The solution containing the acid-soluble ATP and Ap4A appeared in overlapping fractions. The ATP/ nucleotides, to which unlabeled carriers of ADP, ATP, GTP, ADP ratios were calculated, based on either 3H- or 32P radio- and Ap4A had been added at the beginning of the extraction activity, which yielded similar values. Values of 10-13 were procedure, was applied on top of a DEAE-Sephadex A-25 col- obtained for the ATP/ADP ratios under normal conditions for umn and eluted with a linear gradient of 0.1-0.6 M NaCI in 0.05 most of the monolayer cell lines. Total cellular ATP was cal- M Tris-HC1, 0.002 M EDTA, and 7 M urea, pH 7.6. Fractions culated by converting the 32p counts in the ATP fraction into containing ATP and Ap4A, which are not separated from each nanomoles. This conversion can be achieved by knowledge of other on this column (both compounds possess a -4 charge), the specific activity of the 32p; in the growth medium, which were pooled, diluted with water to 250 ml, and applied on top in turn is calculated from the known phosphate concentration of DEAE-cellulose-bicarbonate column. Elution with a linear (generally 1 mM) in the medium and the 32P radioactivity of gradient of 0.0-0.6 M ammonium bicarbonate (pH 8.2) af- the medium. This method is based on the assumption that the forded ample separation of ATP and Ap4A. The fractions 32P, in the medium completely equilibrates with the cellular containing the Ap4A were pooled, and the buffer was removed 32P, within 6 hr, reaching similar specific activities. Experi- by repeated evaporation and lyophilization. The dry residue mental evidence supports this assumption (22). Total cellular was dissolved in 50 gII of water, and part of the solution was ATP is thus expressed in nmol/mg of protein or nmol/106 cells, subjected to alkaline phosphatase or snake venom phosphodi- and the total Ap4A is calculated from the Ap4A/ATP ratios of esterase digestion followed by thin-layer chromatography on either the 3H- or 32P-labeled compounds, corrected by factors PEI-cellulose. Elution was performed with water followed by of ½ or %4, respectively. 1 M LiCl. The plate was cut and material was eluted from the Fractions containing the ATP and Ap4A were pooled from small pieces with 4 M ammonium hydroxide for at least 1 hr the DEAE-Sephadex-7 M urea column, and chromatographed before the radioactivity of the eluate was determined in Bray's on a DEAE-cellulose column (Fig. 2), where adequate sepa- scintillation fluid. Alkaline phosphatase digestion was per- ration was achieved due to the extra retardation of Ap4A caused formed in 0.1 M Tris-HC1, pH 8.0, at 370 for 1 hr; snake venom by the interaction of its two adenine moieties with the column Downloaded by guest on September 26, 2021 3986 Biochemistry: Rapaport and Zamecnik Proc. Natl. Acad. Sci. USA 73 (1976) Table 1. Ap4A/ATP ratios obtained by radioactive labeling of acid-soluble nucleotides with [3HI adenine and 32p, Approximate doubling time Cell line (hr) Ap4A/ATP* Cellular ATP level Py-BHK 21/13t 11-13 8-15 X 10-4 5-6 nmol/106 cells, 15-20 nmol/mg of protein BHK 21/13t 12-14 5-10 x 10-4 5-6 nmol/106 cells, 15-20 nmol/mg of protein C3A, human hepatoma (in mono- layer culture)t 12-14 5-8 X 10-4 C3A, human hepatoma [in the nude mouse (21)] 3-4 x 10 4 3mM Polyoma-transformed NILt 12-14 2-3 X 10- 12-14 nmol/mg of protein NILt § 16 0.5 X 10-4 12 nmol/mg of protein CEF1 0.3-0.5 X 10-4 CHOII 18-20 0.2-0.3 X 10-4 20-22 nmol/mg of protein Normal mouse liver 0.1-0.2 x 10-4 3 mM

* Ratios were calculated from the 3H cpm values after correction of the Ap4A values by a factor of0.5. t Kindly supplied by Ms. Maureen Gammon. T Initiated and kindly furnished by Ms. Gammon. § Hamster fibroblast, NIL strain. ¶ Second transplant of primary explant of chick embryo fibroblasts. II Chinese hamster ovary cells, kindly supplied by Dr. Arthur Pardee.

matrix. Further purification of Ap4A was necessary in most (Py-) BHK cells, and the human hepatoma cell line C3A, the cases and was achieved by desalting the pooled Ap4A fractions, Ap4A fractions obtained from the DEAE-cellulose columns followed by alkaline phosphatase digestion (to which Ap4A is were more than 90% pure. In all other cell lines and in normal resistant) and thin-layer chromatography on PEI-cellulose mouse liver, the Ap4A fraction from the DEAE-cellulose col- plates (Fig. 3a). The level of Ap4A was calculated from its ra- umn included an unidentified compound that was susceptible dioactivity in the DEAE-cellulose column, and was corrected to alkaline phosphatase digestion and that accounted for most by the ratio of the counts remaining in the Ap4A fraction after of the radioactivity in this fraction. Normal mouse liver and alkaline phosphatase digestion to the total radioactivity on the subcutaneously grown human hepatoma in the nude mouse thin-layer plate. Extensive digestion of the Ap4A fraction with were labeled in vivo with [3H]adenine and 32P, as described snake venom phosphodiesterase yielded the radioactivity in the (21). AMP fraction produced from the unlabeled carrier (Fig. 3b), Values of Ap4A relative to the total cellular ATP, which were thus affording further proof of the equivalence of the ra- calculated from [3H]Ap4A (corrected by a factor of 0.5) and dioactively labeled material with the added unlabeled car- [3H]ATP, for a variety of cells are listed in Table 1. The rier. Ap4A/ATP ratio thus seems to be inversely related to the For baby hamster kidney (BHK) cells, polyoma-transformed doubling time of the cells, and is not related to the total cellular ATP level under conditions where the ATP/ADP ratio is above Ap4A ADENOSINE 10. The values reported in Table 1 include only intracellular CD Ap4A. The extensive washing procedure before the extraction of the intracellular acid-soluble nucleotides eliminates the possible presence of any extracellular Ap4A. Moreover, Ap4A was unstable in medium containing 15% unheated fetal calf serum. The possibility of some of the Ap4A being formed arti- factually due to a reaction of ATP under the isolation or sepa- ration conditions was eliminated by carrying comparable AMP ADENOSINE amounts of radioactively labeled ATP through the isolation and ( separation procedures without detection of any Ap4A. 10r2- Variation of Cellular Ap4A Levels with Degree of Prolif- 6 erative Activity of Cells. The human malignant hepatoma cell 4 line (C3A), which we grew subcutaneously in the nude (athy- mic) mouse, yielded Ap4A/ATP ratios of 3 to 4 X 10-4 after acid-soluble nucleotides were labeled in vivo with [3H]adenine 5 10 15 20 and 32Pi by techniques reported earlier (21). The liver of the DISTANCE FROM OR/GIN (cm) same mouse yielded Ap4A/ATP ratios of 0.1 to 0.2 X 10-4 FIG. 3. Thin-layer chromatography of fractions 73-76 from the under conditions where the ATP/ADP ratio was 2-3. Both the DEAE-cellulose column described in Fig. 2 on PEI-cellulose plates, liver and tumor had a total ATP level of 2-3 mM based on wet after removal of the volatile buffer by evaporation and lyophilization. tissue weight (21). The Ap4A level in the human hepatoma cells Elution was performed with water followed by 1 M LiCl. (a) Sample treated with alkaline phosphatase. (b) Sample treated with snake grown subcutaneously in the nude mouse is therefore 0.8 to 1.2 venom phosphodiesterase. Unlabeled adenosine was added before ,uM, while the Ap4A level in the normal mouse liver is 0.03-0.06 chromatography. JM. Downloaded by guest on September 26, 2021 Biochemistry: Rapaport and Zamecnik Proc. Natl. Acad. Sci. USA 73 (1976) 3987 BHK cells yield an Ap4A/ATP ratio of 5 to 10 X 10-4; how- Table 2. Effect of rapid partial depletion of cellular ATP ever, these BHK cells, arrested in the G1 phase of the cell cycle pool on levels of Ap4A* by serum deprivation, total amino acid starvation, or isoleucine deprivation (23, 24), produce a substantial and consistent drop Cell line ATP/ADPt Ap4A/ATPt of 30- to 50-fold in their Ap4A levels, yielding Ap4A/ATP ratios BHK 16.4 13.2 x 10-4 of 0.1 to 0.2 X 10-4. The total steady-state levels of ATP in BHK BHK 12.1 2.0 x 10-4 cells is not more than 2-fold higher than the steady state levels BHK 8.3 1.6 x 10-4 of ATP in GI-arrested BHK cells, with the normal steady-state BHK 7.2 1.2 x 10-4 level of cellular ATP at 15-20 nmol/mg of protein. The ATP/ Py-BHK 12.8 9.9 x 10-4 ADP ratios in asynchronized BHK cells and BHK cells arrested Py-BHK 11.4 6.3 x 10-4 in the GI phase of the cell cycle are also similar. BHK cells ar- Py-BHK 10.6 4.7 X 10-4 rested in the GI phase of the cell cycle by either one of the three Py-BHK 6.3 4.7 X 10-4 procedures used, produced a 10- to 20-fold decrease in 1 hr of * Cells were grown in 55-cm2 petri dishes. After removal of the [3H]thymidine incorporation as compared with normally medium, the cells were exposed for various lengths of time before growing BHK cells. the addition of ice-cold 0.5 M perchloric acid. Longer exposure A large dose (2 X 107) of BHK cells, when injected subcuta- times resulted in lower ATP/ADP ratios. t Similar values were obtained from the 3H ratios and the 32p ratios neously into two nu/nu mice (kindly furnished by Dr. Wendall corrected by 2/3. Farrow, Life Sciences Laboratories, St. Petersburg, Fla., $ Ratios were calculated from the 3H cpm values after correction of through the auspices of Dr. Jack Gruber of the Virus Cancer the Ap4A values by a factor of0.5. Program) produced no tumor in one mouse and a small tumor nodule in the second mouse, which remained quiescent for 3 weeks, then abruptly grew to large proportions, eventually DISCUSSION killing this mouse. A similar number of Py-BHK cells grew The presence of Ap4A in a variety of mammalian cells in a live immediately subcutaneously, and killed all of a series of four animal or in tissue cultures has been demonstrated. This com- nu/nu mice by the enormous size of the tumor and local in- pound, which was previously shown to be formed in vitro in volvement within 3 weeks after injection. In our experience, the backreaction of the amino acid activation process (Eq. 2), similar large doses of recently explanted normal cells have never was detected in concentrations ranging from as high as 0.8-1.2 produced tumors in this nu/nu mouse. We therefore regard ,uM in human hepatoma grown subcutaneously in the nude the variant of the BHK line with which we have been dealing mouse, to 0.03-0.06 AuM in normal mouse or rat liver. In mo- as a neoplastic cell, of low in vivo invasive potential in com- nolayer cultures, Ap4A was detected in concentrations ranging parison with the Py-BHK cell line, yet still retaining growth from 30 pmol/mg of protein in cells with a high rate of division restraints in tissue culture generally associated with nonma- to 0.15 pmol/mg of protein in slowly growing cells with a low lignant cells. division rate. These values can also be expressed as 15 X 10-4 Protein synthesis was inhibitied in BHK cells by the use of to 0.1 X 10-4 of the steady-state levels of cellular ATP, re- puromycin (100 ttg/ml of medium) or pactamycin (10 ug/ml) spectively. The steady-state level of ATP, which does not reflect for 18 hr. The uptake of [3H]leucine into acid-insoluble material the rate of turnover of ATP, was found to be unrelated to the declined 200- to 300-fold compared with normal BHK cells. proliferative activity of the tissue. The level of Ap4A, however, The Ap4A/ATP ratios in BHK cells treated with puromycin is directly related to the proliferative activities of the cells as- or pactamycin declined at least a 100-fold, to 0.1 X 10-4. The sayed for its presence. total steady-state level of ATP did not, however, significantly The metabolic lability of Ap4A was higher than the metabolic change (10-15 nmol/mg of protein), and, in addition, the cel- lability of ATP under certain conditions. In an individual cell lular ATP/ADP ratios remained at the high level of 10-15. line, the Ap4A level dropped with a decrease in the ATP/ADP BHK and polyoma-transformed BHK cells were grown for ratio. A decrease in the ATP/ADP ratio was caused by a nu- 48 hr in Dulbecco's modified Eagle's medium supplemented tritional shiftdown, which was produced by allowing longer with 15 or 0.25% fetal calf serum. Growth of BHK cells was periods of time to elapse between removal of the medium and slowed in medium containing low serum concentrations, as fixation of the cells with perchloric acid. It is thus suggested that indicated by [3H]thymidine incorporation or cell count, while Ap4A may serve as a backup system for ATP for some cellular the growth of Py-BHK cells was slowed to a smaller degree in functions, and is utilized very rapidly under conditions in which low serum. In the medium supplemented with high serum the ATP pool is rapidly depleted. Another possibility is rapid concentration, BHK and Py-BHK cells divided every 13 and enzymatic degradation of Ap4A (19), once such conditions have 12 hr, respectively, as was indicated by cell counts. While the been introduced. total steady-state levels of cellular ATP and the ATP/ADP ratios The results obtained in this study indicate that Ap4A is as- did not significantly change in going from high to limited serum sociated with cellular proliferation. Its low concentration and concentrations, the total cellular Ap4A levels dropped 20-fold high metabolic lability, however, may suggest a function such in BHK and only 10-fold in Py-BHK cells. as that of a "signal nucleotide" or a positive "pleiotypic acti- The metabolic lability of Ap4A as compared with that of ATP vator." The observations of up to a 50-fold difference in the is illustrated in Table 2. Short periods of nutritional shiftdown concentration of Ap4A in going from normally growing asyn- produced by removal of the medium from the cellular mono- chronized cells in culture to cells arrested in the GI phase of the layer yield a decrease in the cellular ATP level, which is also cell cycle further strengthen the functional possibility of Ap4A expressed by a lower ATP/ADP ratio. The intracellular Ap4A as a "signal nucleotide". BHK cells were arrested in the GI levels drop at least as fast as the intracellular ATP levels, as is phase of the cell cycle by serum deprivation or amino acid shown by lower Ap4A/ATP ratios after the sudden nutritional starvation for 48 hr (23, 24). Although a decrease of up to 50- shiftdown. Relative metabolic stability of Ap4A would have fold was observed in the Ap4A levels under such conditions, the produced higher Ap4A/ATP ratios after a partial depletion of steady-state level of cellular ATP was not altered more than the ATP pool. 2-fold. Cellular proliferation of BHK cells was also arrested by Downloaded by guest on September 26, 2021 3988 Biochemistry: Rapaport and Zamecnik Proc. Natl. Acad. Sci. USA 73 (1976) 18 hr of incubation in the presence of puromycin, which inhibits [E(11-1)2404]. E.R. is indebted to Aid for Cancer Research of Brook- protein synthesis by releasing incomplete peptide chains from line, Mass., for a fellowship. ribosomes (25), or in the presence of pactamycin, which inhibits protein synthesis by inhibiting interactions between ribosomes, 1. Tomkins, G. M. (1975) Science 189,760-763. mRNA, and tRNA (26). Under these conditions the cellular 2. Sutherland, E. M. (1972) Science 177,401-408. Ap4A levels dropped more than a 100-fold, suggesting a link 3. Abell, C. W. & Monahan, T. M. (1973) J. Cell Biol. 59, 549- between the rate of protein synthesis and intracellular Ap4A 558. levels. We presently assume, without proof, that the synthesis 4. Pastan, I. H., Johnson, G. S. & Anderson, W. B. (1975) Annu. Rev. of Ap4A in vivo proceeds through the backreaction of the amino Biochem. 44, 491-522. 5. Cashel, M. & Gallant, J. (1969) Nature 221, 838-841. acid activation process, which was demonstrated by the in vitro 6. Rapaport, E. & Bucher, N. L. R. (1976) in Onco-Developmental studies ( 1-15). The level of Ap4A can thus be controlled by a Gene Expression, eds. Fishman, W. H. & Sell, S. (Academic Press, combination of its rate of synthesis (11-15) nd rate of degra- New York), in press. dation (19). Normal adult mouse liver, which is a Go-arrested 7. Mamont, P., Hershko, A., Kram, R., Schacter, L., Lust, J. & tissue with a negligible cell division rate, yields a low level of Tomkins, G. M. (1972) Biochem. Biophys. Res. Commun. 48, Ap4A (0.03-0.06 ,uM) despite its considerable rate of protein 1378-1384. synthesis. In liver the Ap4A levels are probably controlled 8. Hershko, A., Mammont, P., Shields, R. & Tomkins, G. M. (1971) mainly by its enzymatic degradation (19). Nature New Biol. 232, 206-211. The data reported here do not rule out the possibility of Ap4A 9. Hoagland, M. B. (1955) Biochim. Biophys. Acta 16,288-289. in 10. Hoagland, M. B., Keller, E. B. & Zamecnik, P. C. (1956) J. Biol. being tissue-specific to a certain degree; however, the same Chem. 218, 345-358. type of cells (e.g., BHK) the intracellular concentration of Ap4A 11. Zamecnik, P. C., Stephenson, M. L., Janeway, C. M. & Randerath, fluctuates widely with the proliferative activity of the cells. K. (1966) Biochem.'Biophys. Res. Commun. 24,91-97. The intracellular concentration of Ap4A was calculated based 12. Randerath, K., Janeway, C. M., Stephenson, M. L. & Zamecnik, on its product-precursor.relationship with ATP. The double P. C. (1966) Biochem. Biophys. Res. Commun. 24,98-105. radioactive labeling of ATP, which uses 32Pi and [3H]adenine, 13. Zamecnik, P. C. & Stephenson, M. L. (1969) Alfred Benzon provides information as to the levels of ATP produced by the Symposium I, The Role of Nucleotides for the Function and de novo synthesis and by the purine salvage pathway (27). Conformation of Enzymes, eds. Kalckar, H. M., Klenow, H., Whereas 32Pi radioactively labels the total ATP, [3H]adenine Munch-Petersen, G., Ottesen, M. & Thuysen, J. M. (Munksgaard, is incorporated mostly into the ATP fraction produced via the Copenhagen), pp. 276-291. 14. Zamecnik, P. C. & Stephenson, M. L. (1968) in Regulatory purine salvage pathway. In BHK cells arrested by serum de- Mechanisms for Protein Synthesis in Mammalian Cells, eds. privation or amino acid starvation, incorporation of [3H]adenine San Pietro, A., Lamborg, M. R. & Kenney, F. T. (Academic Press, into ATP is enhanced relative to the fraction of ATP that in- New York), pp. 3-16. corporates [3H]adenine in normally growing BHK cells. A few 15. Zamecnik, P. C. (1969) Cold Spring Harbor Symp. Quant. Biol. experiments have suggested that Ap4A in normally growing 34, 1-16. BHK cells is produced from a special pool of compartmental- 16. Rapaport, E., Svihovec, S. K. & Zamecnik, P. C. (1975) Proc. Natl. ized ATP consisting more of purine salvage product than of Acad. Sci. USA 72,2653-2657. ATP synthesized de novo (32P/3H ratios in Ap4A are smaller 17. Finamore, F. J. & Warner, A. H. (1963) J. Biol. Chem. 238, than 32P/3H ratios of ADP or % X 32P/3H ratios of ATP, indi- 344-348. 18. Warner, A. H. & Huang, F. C. (1974) Can. J. Biochem. 52, cating preferential incorporation of [3H]adenine into Ap4A). 241-251. This specific point is still unresolved. An experiment in which 19. Vallejo, C. G., Lobaton, C. D., Quintanilla, M., Sillero, A. & Sil- [14C]glycine was used to label mostly the ATP produced de lero, M. A. G. (1976) Biochim. Biophys. Acta 438,304-309. novo yielded somewhat lower levels of radioactive label in 20. Reiss, J. R. & Moffatt, J. G. (1965) J. Org. Chem. 30, 3381- Ap4A but only by a factor of 2-3. 3387. The sharp and dramatic shifts in the intracellular concen- 21. Rapaport, E. & Zamecnik, P. C. (1976) Proc. Natl. Acad. Sci. USA tration of Ap4A that are associated with the cellular proliferative 73,3122-3125. activities, as well as the metabolic lability of Ap4A, suggest that 22. Colby, C. & Edlin, G. (1970) Biochemistry 9,917-920. this nucleotide acts as a signal for a positive pleiotypic re- 23. Clarke, G. D., Stoker, M. G. P., Ludlow, A. & Thornton, M. (1970) at onset of cor- Nature 227,798-801. sponse-a signal formed the protein synthesis 24. Holley, R. W. & Kiernan, J. A. (1974) Proc. Natl. Acad. Sci. USA relating its rate to other cellular functions that determine the 71,2942-2945. rate of cell division. 25. Allen, D. W. & Zamecnik, P. C. (1962) Biochim. Biophys. Acta We acknowledge the expert technical assistance of Mrs. Mary Za- 55,865-875. mecnik and Ms. Sandra Svihovec. This is Publication no. 1506 of the 26. Felicetti, L., Colombo, B. & Baglioni, C. (1966) Biochim. Biophys. Cancer Commission of Harvard University, supported by National Acta 119, 120-129. Cancer Institute Virus Cancer Program (NO1 CP-33-66) and the United 27. Murray, A. W., Elliott, D. C. & Atkinson, M. R. (1970) Prog. States Energy Research and Development Administration Contract Nucleic Acid Res. Mol. Biol. 10, 87-119. Downloaded by guest on September 26, 2021