effective adaptive mechanisms. This hypothesis is supported by previous observations as well. Macroplasmodia main- tained protoplasmic streaming for up to ...
JOURNAL OF BACTERIOLOGY, Mar. 1989, P. 1506-1512 0021-9193/89/031506-07$02.00/0 Copyright © 1989, American Society for Microbiology
Vol. 171, No. 3
Changes in Diadenosine Tetraphosphate Levels in Physarum polycephalum with Different Oxygen Concentrations PRESTON N. GARRISON, SANDRA A. MATHIS, AND LARRY D. BARNES* Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760 Received 15 July 1988/Accepted 8 December 1988
Cellular levels of diadenosine tetraphosphate (Ap4A) were measured, by a specific high-pressure liquid chromatography method, in microplasmodia of Physarum polycephalum subjected to different degrees of hypoxia, hyperoxia, and treatment with H202. Ap,A levels increased three- to sevenfold under anaerobic conditions, and the microplasmodia remained viable after such treatment. Elevated levels of Ap4A returned to the basal level within 5 to 10 min upon reoxygenation of the microplasmodia. The increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in fed, log-phase microplasmodia. The maximal increase measured in log-phase microplasmodia was twofold. No significant changes in Ap4A levels occurred in microplasmodia subjected to mild hypoxia, hyperoxia, or treatment with 1 mM H202. These results indicate that in P. polycephalum, Ap4A may function in the metabolic response to anaerobic conditions rather than in the response to oxidative stress.
Diadenosine 5',5' "-P',P4-tetraphosphate (Ap4A) was discovered by Zamecnik and co-workers during experimentation on in vitro catalysis by lysyl-tRNA synthetase from Escherichia coli (42, 51). On the basis of their subsequent studies, Zamecnik and co-workers proposed that Ap4A may serve as a regulatory nucleotide that couples protein synthesis and replication or transcription (44, 50). Studies supportive of Ap4A involvement in DNA replication have been reviewed (6, 18, 49). However, Bambara et al. (5) have presented arguments against this putative function of Ap4A. The constancy of Ap4A during traverse of the cell cycle in Physarum polycephalum (16), E. coli (40), and cultured mammalian cells (35, 38) also argues against the hypothesis that Ap4A is a positive regulator of DNA synthesis. A second hypothesis is that Ap4A and related dinucleoside polyphosphates are signal nucleotides or alarmones of oxidative stress in procaryotic organisms (8, 26, 27). This hypothesis is based on the marked increase in Ap4A and other dinucleoside polyphosphate levels in E. coli and Salmonella typhimurium upon treatment with aromatic oxidants, ethanol, sulfhydryl reagents, agents that decrease glutathione, and heat shock (8, 26, 27). Oxidative stress was proposed to be the common element among these different conditions and reagents (8). A comparison of the levels of dinucleoside polyphosphates in E. coli subjected to conditions that affect the heat shock, oxidative stress, and SOS regulons indicated that a consistent role for these nucleotides as alarmones was associated only with the response to oxidative stress (48). Recently, culture conditions and agents commonly associated with induction of stress proteins have been shown to increase Ap4A, diadenosine 5'5'"'-Pl,P3-triphosphate (Ap3A), or adenosine 5',5'''-P1,P4-tetraphosphoguanosine (Ap4G) or more than one of these dinucleotides in eucaryotic organisms. Heat shock treatment increased Ap3A in yeast cells (13) and increased both Ap4A and Ap3A in Artemia sp. (33). In cultured Drosophila cells, levels of Ap4A, Ap3A, and Ap4G were increased by mild heat shock and 5 mM CdCI2(9). Several stress conditions and agents, including CdCl2, ethanol, and heat shock, that induce the synthesis of stress proteins increased the level of adenyly*
lated nucleotides in cultured mouse cells (3). Ap4A and Ap4G increased in P. polycephalum macroplasmodia treated with dinitrophenol, and the increases were larger in starved than in fed macroplasmodia (16). However, on the basis of negligible changes in Ap4A in 3T3 and hamster lung cells subjected to heat shock or treatment with paraquat, duroquinone, platinum compounds, or ethanol, Segal and LePecq (46) suggested that Ap4A was not an alarmone of oxidative stress in eucaryotic cells. In yeast cells subjected to heat at 46°C or 1 mM cadmium acetate, the Ap4A level increased about 50-fold, but these treatments resulted in cell death (4). Similarly, accumulation of Ap4A in Xenopus laevis and cultured hepatoma cells treated at 45°C was associated with cell death, and there was no apparent correlation between the synthesis of heat shock proteins and the increased levels of dinucleoside polyphosphates (20). Here we describe the measurement of Ap4A in P. polycephalum microplasmodia subjected to different concentrations of oxygen and H202. Significant increases in Ap4A levels occurred during anaerobiosis, and the increases were rapidly reversed upon reoxygenation. These increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in log-phase microplasmodia. Physarum microplasmodia were still viable after these treatments. Only small changes occurred during hyperoxia or treatment with H202. Thus, Ap4A is probably not an alarmone of oxidative stress in P. polycephalum but it may be a signal nucleotide for metabolic responses to anoxic conditions. (A preliminary report of this work has been presented in an abstract [L. D. Barnes, S. A. Mathis, and P. N. Garrison, Fed. Proc. 46:2217, 1987]. ) MATERIALS AND METHODS Materials. Ap4A was purchased from Sigma Chemical Co. (St. Louis, Mo.). It was custom labeled with 3H by Amersham Corp. (Arlington Heights, Ill.) and was purified by chromatography on dihydroxyboronyl-BioRex 70 (7). Partisil 10 SAX columns, high-pressure liquid chromatography resin, and DEAE-cellulose were obtained from Whatman, Inc. (Clifton, N.J.). E. coli alkaline phosphatase, horseradish peroxidase, catalase, H202, 4-aminophenazone, and 4,5-
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dihydroxynaphthalene-2,7-disulfonic acid were purchased from Sigma. Ar, N2, and 02 were from Liquid Carbonic. V205 was from Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Culture conditions. P. polycephalum M3CVII cells were grown as microplasmodia in gyratory shake culture at 150 rpm and 25 to 26°C in semidefined medium (11) as previously described (15). Microplasmodia were used in log phase (2 to 3 g [wet weight]/100 ml of culture) or early stationary phase (near the peak culture density of 10 to 12 g [wet weight]/100 ml) or transferred at log phase to a starvation medium for various periods of time. The starvation medium was spherulation salts (11) or a medium containing salts, biotin, thiamine, and tryptone but lacking glucose (12). It was necessary to concentrate all cultures except those in stationary phase to 10 to 12 g (wet weight)/100 ml (in the conditioned medium) before use to keep sample volume manageable. This was done for log-phase cultures 2 h before use and for starvation cultures at the time of transfer to starvation medium. Treatment with hypoxic conditions or H202. In some experiments, hypoxia was induced in microplasmodia in the various media by transferring 6-ml portions of culture to 50-ml conical tubes, centrifuging the tubes at 140 x g for 20 s, and allowing the plasmodial pellets to stand under the medium in a 25°C incubator for various times. Samples were collected by adding 0.7 to 0.8 ml of 50% (wt/vol) trichloroacetic acid (CC13COOH) or 60% (wt/vol) perchloric acid (HC104), each containing 1 pmol of [3H]Ap4A, vortexing the samples, and placing them on ice. Some samples were reaerated by being transferred to 50-ml flasks and returned to the shaker. They were collected by direct addition of acid as described above. Hypoxic and hyperoxic conditions were also induced by continuous flushing of a shaking culture flask (500-ml baffled flask with 100 ml of medium) with 02, N2, an N2-02 mixture, or Ar. N2-02 mixtures were preadjusted with shaking medium. When Ar was used, it was sparged through 20 mM V(II) in H2SO4 over Zn (32), followed by 0.1 M potassium citrate (pH 5). For reaeration, the flask was flushed with a stream of air. Dissolved 02 was monitored with a model 53 02 monitor (Yellow Springs Instrument Co., Yellow Springs, Ohio) and a Clark electrode inserted through the flask stopper or in a horizontal port near the base of the flask. Samples (6 ml) were removed with a syringe attached to a sampling-vent tube, which was pushed into the medium only during sampling. Positive gas pressure enabled sampling to be done without introduction of any air. Samples were acid fixed as described above. Treatment with H202 was done by slowly adding appropriate amounts of 1 M H202 to a shaking culture (100 ml in a 500-ml flask). The concentration of stock H202 was determined by using E240 = 43.6 M-1 cm-1 (31). Samples (6 ml) were acid treated as described above. Preparation of acid-soluble extracts. After microplasmodia were collected, the acidified samples were immediately sonicated by using a microtip on a Heat-Systems-Ultrasonics W-375 unit. Two 30-s sonication periods on ice were separated by 60 s of cooling on ice. After 30 min on ice, samples were centrifuged at 31,000 x g for 45 min at 4°C. Samples in 5% CC13COOH were adjusted to pH 6 with NH40H before storage at -20°C. Sample fractionation. Samples containing glucose (log phase), which interferes with boronate chromatography, were collected in HC104. To these samples, 10 ,ul of CH3COOH was added, and sufficient 10 N KOH was added
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dropwise during vortexing to bring the pH to 4 to 6. After 15 min on ice, KC104 was removed by centrifugation, and the supernatants were diluted 1:1 with H20. The samples were then applied to 0.8-by-5-cm columns of DEAE-cellulose equilibrated with 20 mM ammonium acetate (pH 6). Washes with 5 ml of 20 mM ammonium acetate (pH 6) and 10 ml of 250 mM ammonium acetate (pH 6) were discarded, and a dinucleotide fraction was eluted with 10 ml of 1 M ammonium acetate (pH 6). This fraction and the samples in 5% CC13CCOH were then adjusted to pH 9.8 with NH40H, ethanol was added to 60%, and the samples were purified by boronate chromatography as previously described (15). Assay of Ap4A and Ap4G. Ap4A and Ap4G were quantitatively assayed by isocratic anion-exchange high-pressure liquid chromatography as previously described (15) except for a modification in the buffer (16). Sample recoveries for Ap4A were 55 to 65% except for 10% lower recoveries for samples that were subjected to DEAE-cellulose chromatography. Recoveries were based on collection of [3H]Ap4A eluates from the high-pressure liquid chromatography column. Recoveries of Ap4G were assumed to equal those of Ap4A. Assay of H202. Samples for determination of H202 (2 to 3 ml of culture or conditioned medium or of plasmodial pellets) were brought to 6% HC104, vortexed, and placed on ice for 30 min. After centrifugation, the supernatant was buffered with 0.15 M K2HPO4 and neutralized with 10 N KOH, and KCl04 was removed by centrifugation. Samples were assayed for H202 by a peroxidase procedure (31). We found that extracts from P. polycephalum contained catalaseinsensitive substances that gave a large positive response in the assay. Consequently, we split each sample and assayed with and without catalase pretreatment to obtain the response due to H202. Viability determination. Quantitative determination of viability in P. polycephalum is made difficult by the syncytial nature of the plasmodia, a large variation in their size in shaken culture, and their ability to seal off damaged regions (11, 21). We have assessed killing by transferring equal amounts of control and treated microplasmodia to growth medium and determining the excess delay to the time of peak growth in the treated sample as a measure of the viable plasmodial mass remaining. The degree of killing can be estimated by assuming a normal shake culture doubling time of about 16 h (determined by protein assay) and allowing for some treatment-induced physiological lag. We have assumed that an excess delay of 2 days or more represents substantial
killing. Protein determination. Acid-insoluble pellets were washed twice with 95% (vol/vol) ethanol, dissolved in 2 N NaOH, and assayed for protein (29), with bovine serum albumin as the standard. Data analyses. Data values represent the means and ranges of duplicate plasmodial samples at each point or concentration. RESULTS Measurement of Ap4A and Ap4G in P. polycephalum microplasmodia under different 02 concentrations. In initial experiments measuring Ap4A in P. polycephalum microplasmodia, we found that higher values were obtained when culture samples were centrifuged than when medium and plasmodia were acidified and collected together (results not shown). This effect was accentuated in starving cultures. To pursue this observation, the dinucleotides were measured at
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