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Presence of Diadenosine 5',5"'-Pl,P4-Tetraphosphate (Ap4a) In

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Presence of Diadenosine 5',5 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.
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