Proc. Nati. Acad. Sci. USA
Vol. 76, No. 8, pp. 3660-3664, August 1979 Biochemistry
Mechanism of toxicity of putrescine in Anacystis nidulans (polyamines/cyanobacteria/spermidine/ribosomes)
LINDA A. GUARINO* AND SEYMOUR S. COHEN Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794
Contributed by Seymour S. Cohen, May 7, 1979
Putrescine is lethal to the cyanobacterium ABSTRACT Anacystis nidulans at extracellular pH values at which significant concentrations of the nonprotonated diamine rapidly diffuse into the cell and accumulate as the charged form. Although over 98% of the accumulated putrescine is not metabolized, a small fraction is rendered trichloroacetic acid-insoluble, and about 90% of this is bound as putrescine to proteins and cell structures. Various synthetic functions were studied in the presence of a bacteriostatic (40 pM) and a bacteriocidal (150 pM) concentration of putrescine at pH 9.5. Under lethal conditions, protein synthesis was completely inhibited after 45 min and CO2 fixation after 100 min, whereas nucleic acid synthesis was less affected. Spermidine was lost from the cell and its synthesis was arrested. These functions were much less inhibited at 40 pM putrescine. Ribosomes from putrescine-killed cells were found to be irreversibly dissociated into 30S and 50S subunits. Some putrescine (1-4 molecules) cosedimented with each subunit.
Putrescine is quite inhibitory to growth of the cyanobacterium Anacystis nidulans (1). The exogenous metabolite is toxic at the concentration (0.15 mM) at which putrescine is normally present within the organism. It appears that uncharged molecules of putrescine diffuse across the cell membrane and are protonated and trapped inside the cells (2). The pH gradient that exists across the cell membrane of these organisms growing under alkaline conditions permits a 500- to 3000-fold concentration of putrescine within the cells (2). The rapid uptake of putrescine to very high intracellular concentrations results in cell death. This paper presents data on the mechanism of this lethal effect. MATERIALS AND METHODS
Uptake of Putrescine and Viability of A. nidulans. A. nidulans strain 625 was grown as described (1). Exponential phase cultures were harvested by centrifugation at 10,000 X g for 5 min, washed, and resuspended at 1 X 108 cells per ml in Allen's medium containing 30 mM NaHCO3 and adjusted to the indicated pH values with NaOH. Cell number was determined in a Petroff-Hauser microscopic counting chamber. Uptake was initiated by the addition of [3H]putrescine at the indicated concentrations at specific activities of 16 or 62.5 ,tCi/,umol (1 Ci = 3.7 X 1010 becquerels). At various times aliquots were collected on Millipore HA filters and washed with water as described (2). Viability was determined by the single cell plating technique (3). Polyamine Assay. The procedures for dansylation of cell
extracts and fluorometric estimation of the dansyl polyamines after separation by thin-layer chromatography have been de-
scribed (1). Radioisotopes and Chemicals. [2,3-3H]Putrescine (20.64 Ci/mmol), [1,4-14C]putrescine (89.9 mCi/mmol), [tetramethylene-1,4-14C]spermidine (71.1 mCi/mmol), [2-14C]uracil The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
(45 mCi/mmol), [8-'4C]deoxyadenosine (38.8 mCi/mmol), [U-14C]leucine (45 mCi/mmol), sodium [14C]bicarbonate (9.3 mCi/mmol), and Liquifluor were purchased from New England Nuclear. Putrescine dihydrochloride, spermidine trihydrochloride, dansyl chloride, and proline were purchased from Calbiochem. Quantagram LQ6D thin-layer chromatography plates were obtained from Kontes. Synthesis of Macromolecules and CO2 Fixation. Cultures in exponential growth were harvested, washed, and resuspended at 8 X 107 cells per ml in Allen's medium (pH 9.5). DNA synthesis was assayed by the incorporation of [8-14C]deoxyadenosine (0.325 Atg/ml) (4). RNA synthesis was followed by the incorporation of [2-14C]uracil (0.5 Ag/ml) (5). [14C]Leucine was used to measure protein synthesis. Experiments were initiated by the addition of the appropriate isotope to separate flasks containing 20-ml cultures. At various times 0.5-ml aliquots were removed and added to an equal volume of cold 10% trichloroacetic acid. After 30 min on ice, the samples were filtered onto Whatman GF/C glass fiber filters and washed three times with 5 ml of cold 5% trichloroacetic acid and 5 ml of cold 95% (vol/vol) ethanol. The filters were dried and radioactivities were measured in Liquifluor. To determine the fraction of [2-14C]uracil incorporated into DNA and [814C]deoxyadenosine incorporated into RNA, the acid precipitates were incubated in 0.3 M KOH at 370C for 18 hr, cooled, precipitated with 0.5 M trichloroacetic acid (6), washed with 5% trichloroacetic acid onto filters, dried, and assayed for radioactivity. The RNA and DNA values given in the text include this correction. Free fatty acids and chlorophyll a were removed by extraction with cold acetone/methanol (7:2, vol/vol) and lipids were extracted by boiling in alcohol/ether (1:1). The lipid-free residue was incubated in 0.3 M KOH at 37°C for 18 hr to hydrolyze RNA. After precipitation with 0.6 M trichloroacetic acid the solution was centrifuged to remove the ribonucleotides and the pellet was heated in 0.3 M trichloroacetic acid at 80°C for 15 min. The solution was chilled and centrifuged to separate the protein fraction from the hydrolyzed DNA. Uptake and aminoacylation of ['4C]leucine were analyzed in growing cultures of A. nidulans in the presence of [14C]leucine at 0.5 ,uCi/ml. At 20 min and 60 min after the addition of leucine, 1-ml aliquots were removed and washed twice in fresh growth media. The cell pellet was extracted twice with cold 5% trichloroacetic acid. The cold-acid residue was extracted twice with hot 5% trichloroacetic acid (900C, 15 min) to hydrolyze [14C]leucyl-tRNA and extracts were pooled and assayed for radioactivity. CO2 fixation was measured as described (2). Isolation of Ribosomes. Ribosomes were isolated from 25-ml cultures by modifications of the method of Goldberg and Steitz (7). The cells were collected by centrifugation, washed twice in buffer A [100 mM NH4C1/10 mM Mg(OAc)2/20 mM Tris*
3660
Present address: Biophysics Laboratory, University of Wisconsin, Madison, WI 53706.
Biochemistry:
Guarino and Cohen
Proc. Natl. Acad. Sci. USA 76 (1979)
3661
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HCl (pH 7.5)/0.5 mM EDTA/3 mM mercaptoethanoll, and broken by sonication in buffer A. The resulting suspension was sedimented to remove cell walls and membranes at 37,000 X g, 40C, in the Sorvall SS34 rotor for 30 min; 3.5 ml of the resulting supernatant was layered over 5.0 ml of 1.1 M sucrose in buffer A-1 [50 mM NH4Cl/10 mM Mg(OAc)2/20 mM Tris-HCl (pH 7.5)/0.5 mM EDTA/3 mM mercaptoethanoll and centrifuged for 16 hr, 40C, at 48,000 rpm in a Beckman R 65 rotor. The ribosome pellet was gently resuspended in 0.5 ml of cold buffer A-1. Aliquots (200 jil) were layered over 5 ml of 5-20% sucrose gradients in buffer A-1 and centrifuged in a Beckman SW 50 rotor for 2 hr at 40,000 rpm, 40C (8). The gradients were collected from the top with an ISCO density gradient fractionator model 183, and the absorbance at 254 nm was monitored in an ISCO Model UA-4 absorbance monitor and simultaneously plotted on a Varian A-25 recorder. Ribosomal subunits were isolated in the same manner except that buffer A and A-1 contained 1 mM Mg(OAc)2. The ribosomal subunits were reassociated by adjusting the resuspended ribosomal subunits to 5 mM Mg(OAc)2 and 5 mM spermidine (9) and centrifuging in 5-20% sucrose gradients in buffer A-1 containing 5 mM Mg(OAc)2 and 5 mM spermidine.
RESULTS Polyamine Metabolism. To determine whether putrescine itself is the toxic agent in A. nidulans or whether it must first be metabolized to a toxic compound, Anacystis was incubated in the presence of [3H]putrescine for 14 hr. The amount of radioactively labeled putrescine that had been accumulated by
the cells was monitored by filtration and compared to the amount of putrescine that could be extracted from the cells and
quantitated after dansylation. As shown in Fig. 1A, the concentration of putrescine determined as dansyl putrescine was almost identical with the accumulation of radioactivity, indicating that there was very little metabolism of putrescine. Intracellular putrescine and internal radioactivity were maximal from 2 hr to 4 hr after the addition of putrescine and were then lost from the cells and appeared in the media at approximately the same rate. After 6 hr, total recoverable putrescine decreased gradually, indicating that putrescine was slowly degraded. However, approximately 0.2% of the 3H radioactivity in the cells was recovered in the trichloroacetic acid-insoluble residue
(Fig. 1C). The amount of trichloroacetic acid-precipitable radioactivity increased with time, and 90% of it was found in a lipid-free, nucleic acid-free protein fraction. After acid hydrolysis, the radioactive component was released as dansylatable putrescine. It could be calculated that at 1 hr there were approximately 70,000 trichloroacetic acid-precipitable putrescine molecules per cell. To examine the effect of putrescine on the internal concentration and synthesis of spermidine, a culture of A. nidulans was labeled with 1.4 MiM [14C]spermidine prior to exposure to 150 1AM [3H]putrescine. In the control cells, the concentration of spermidine increased 0.15 nmol/ml per hr for 14 hr (Fig. 1B) and the specific radioactivity of spermidine decreased with a half-life equal to the generation time (10). Under conditions of putrescine toxicity, spermidine increased 0.10 nmol/ml in the first hour, remained constant for another hour, and then was
Biochemistry: Guarino and Cohen
3662
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Biochemistry:
Proc. Nati. Acad. Sci. USA 76 (1979)
Guarino and Cohen
3663
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lost from the cells. The specific radioactivity of spermidine did not decrease after the first hour of incubation in the presence of putrescine, indicating that spermidine synthesis was inhibited. At 40,M putrescine, a nonlethal concentration, spermidine was not lost from the cells. Effect of Putrescine on the Synthesis of Macromolecules. Various synthetic activities, CO2 fixation, and DNA, RNA, and protein synthesis were monitored at two external concentrations of putrescine (40 and 150 MM) and in the absence of external putrescine at pH 9.5 (Fig. 2). The rates of putrescine-accumulation are shown in Fig. 2A. In the 40 MM culture the uptake of putrescine was linear for 120 min, at which time the intracellular concentration was 35 mM. As shown in Fig. 2B, this had a bacteriostatic effect. In the culture treated with 150 ,M putrescine, uptake was linear for 100 min and leveled off at an intracellular concentration equal to 87 mM. After 180 min, the intracellular putrescine slowly began to leak out of the cells. In this culture (Fig. 2B) the cells rapidly lost viability, leaving approximately 700 viable cells per ml. Of the four synthetic processes analyzed, protein synthesis was the first inhibited by putrescine. In the culture incubated in the presence of 150 MM putrescine, protein synthesis was abruptly arrested 35-40 min after addition of the diamine (Fig. 2D). In the 40MM-treated culture, the rate of protein synthesis was decreased 15% compared to the control. The inhibition of protein synthesis was probably not due to an inhibition of uptake or aminoacylation of [14C]leucine, as determined by
30 35 25 20 15 Fraction FIG. 4. The cosedimentation of trichloroacetic acid-precipitable putrescine and ribosomes. Ribosomes were isolated as described in the legend to Fig. 3A. Two-drop fractions were collected onto filters and precipitated in 5% trichloroacetic acid ( --- ). -, UV tracing.
5
10
analysis of intracellular [14C]leucine in cold and hot 5% trichloroacetic acid extracts, respectively. The levels of radioactivity in these fractions were equal to those in the controls. CO2 fixation (Fig. 2C) was not appreciably affected by the presence of 40,uM putrescine throughout the experiment. In the culture treated with 150 MM putrescine, CO2 was fixed at the control rate for 100 min and then the rate of CO2 fixation was decreased to 4% of the control rate. The synthesis of RNA (Fig. 2F) was not affected by 40 MM putrescine, whereas it was inhibited slightly 30 min after the addition of 150 MuM putrescine and slowed down more rapidly after 100 min. The incorporation of [14C]deoxyadenosine appears to be initially increased by the addition of 40 M putrescine (Fig. 2E). This could be due to an effect of putrescine on the permeability of deoxyadenosine, which normally enters the cells very slowly (4). DNA was synthesized in the 150 MuM-culture at a constant rate, which was about 60% of the control rate. Because protein synthesis was inhibited first, the effect of putrescine accumulation on the integrity of the protein synthetic machinery was examined further. Effect of Putrescine on Ribosome Structure. Cells were incubated in the presence or absence of 150,uM putrescine at pH 9.5 for 2 hr. Ribosomes were isolated in the presence of 10 mM Mg2+ and analyzed in sucrose gradients (Fig. 3A). In the control cells, 85% of the ribosomes were present as 70S ribosomes, whereas in the putrescine-treated culture, 96% of the ribosomes were dissociated into 30S and 50S subunits. Ribosomes from control cells isolated in 1. mM Mg2+ dissociated into
Biochemistry: Guarino and Cohen
3664
IntraExternal cellular putrescine, putrescine, mM* AuM 0 1
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0.15 1.2 15.3 68.4 100.1
Proc. Nati. Acad. Sci. USA 76 (1979)
Table 1. Summary of the metabolic effects of putrescine Molecules putrescine per cell Rate of proRibosome Cell walls tein synthesist dissociation,§ + Ribosomal Soluble Lethalityt % inhibition membranes % pellet proteins 1.14
0
9
NA
NA
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0 10 88 96
13.5 23.5 58.0 78.0
85,000 1,359,000 915,000
3,000 46,000 15,000 20,000
891,000
NA 4,000 35,000 68,000
91,000
Molecules putrescine per ribosomal unit 30S 50S 70S NA
NA
NA
0.3 1.5 0.5 0.5
0.3 1.6 0.5 1.0
0.1 0.7 1.1 1.9
NA, not applicable. * Intracellular concentration of putrescine at equilibrium 100-150 min after addition of putrescine. t Lethality expressed as viable count at 120 min/viable count at zero time. Calculated 120 min after addition of putrescine. § Percent of total absorbance present as 30S and 50S subunits, 120 min after addition of putrescine; average of duplicate determinations.
50S and 30S subunits (Fig. 3B), which then readily reassociated into 70S ribosomes in the presence of 5 mM Mg2+ and 5 mM spermidine (Fig. 3C). Ribosomes isolated from putrescinetreated cells in 1 mM Mg2+ were dissociated into several small components (Fig. 3B). In the presence of 5 mM Mg2+ and 5 mM spermidine, they reformed 30S and 50S subunits with a small peak at 70S. The addition of 50 mM putrescine to purified 70S control ribosomes in vitro did not dissociate the ribosomes. A possible relationship between trichloroacetic acid-insoluble putrescine and ribosome dissociation was then examined. Ribosomes were isolated, as described above, from cells that had been incubated in the presence of 150 ,uM [14C]putrescine for 2 hr. As shown in Fig. 4, radioactivity was associated with 30S and 50S subunits. The ribosomes were hydrolyzed with acid (6 M HC1, 110°C, 18 hr) and dansylated. The radioactive dansyl derivative isolated from the ribosomes comigrated with standard dansylputrescine, indicating that putrescine was not metabolized prior to binding to the ribosomes. On the basis of input specific radioactivity (26.6 ,uCi/,umol), extinction coefficients (11), and molecular weights (12) of ribosomes and subunits, there were 2.7 molecules of putrescine per 30S subunit and 4.3 molecules of putrescine per 50S subunit. In a separate experiment, the peak fractions were collected and resedimented through sucrose gradients. The radioactivity due to putrescine comigrated with the ribosome peaks. in the second gradient. In this experiment, there were 1.6 and 3.4 molecules of putrescine per 30S and 50S
subunit, respectively. Summary of the Metabolic Effects of Putrescine. Cultures of A. nidulans were incubated in the presence of the indicated concentrations of [14C]putrescine. Uptake and accumulation of putrescine were monitored continuously for 3 hr. After 2 hr, 25 ml was removed from each culture and ribosomes were isolated. In Table 1, it can be seen that the lethal action of putrescine correlates fairly well with an inhibition of protein synthesis and the dissociation of 70S ribosomes. The distribution of radioactive putrescine recovered in the various cell fractions and in the ribosomal subunits is shown in the last columns. In these columns it would appear that the extent of covalent binding of putrescine to the cell fractions at 2 hr is decreased by cell killing. A clear relation of acid-insoluble ribosomal putrescine to the dissociation of the ribosomes has not been demonstrated. DISCUSSION The addition of toxic concentrations of putrescine to growing cultures of A. nidulans results in a disruption of several metabolic functions within the cells. Complete inhibition of protein synthesis was the earliest event observed after addition of 150
,uM putrescine to the growth medium. This inhibition of protein synthesis was accompanied by an irreversible dissociation of ribosomes. The concentrations of various ions are very important in maintaining intact, functional, 70S ribosomes. In vitro dialysis of purified Escherichia coli 70S ribosomes against spermidine or putrescine can lead to-a stoichiometric replacement of Mg2+ with polyamine, resulting in dissociation (13), irreversible conformational alterations, loss of polymerizing activity, and loss of specific proteins from the 50S subunit (14, 15). Spermidine has also been found to contribute to ribosome stability (9). When A. nidulans was incubated in 150 ,uM putrescine, cellular spermidine was lost from the cell. It is probable that some of the spermidine originally bound to the ribosomes was replaced by putrescine. Putrescine at the high intracellular concentrations observed under conditions of lethality and ribosomal dissociation could compete for various binding sites on the ribosomes, thereby displacing other cations and leading to dissociation. It is possible also that the displacement of essential cations from the ribosomes may also open certain specific sites for covalent binding by putrescine; this possibility of specific inactivation of the ribosomal subunits has yet to be tested. We thank Drs. Dennis Hruby and Richard Condit for helpful discussions. This work was supported by Grant PCM 78-04324 from the National Science Foundation. 1. Ramakrishna, S., Guarino, L. A. & Cohen, S. S. (1978) J. Bacteriol. 134,744-750. 2. Guarino, L. A. & Cohen, S. S. (1979) Proc. Natl. Acad. Sci. USA
76,3184-3188.
3. Allen, M. M. (1968) J. Phycol. 4, 1-4. 4. Restaino, L. & Frampton, E. W. (1975) J. Bacteriol. 124, 155160. 5. Pigott, G. H. & Carr, N. G. (1971) Arch. Microbiol. 79, 1-6. 6. Stern, J. L., Sekiguchi, M., Barner, H. D. & Cohen, S. S. (1964) J. Mol. Biol. 8,629-637. 7. Goldberg, M. L. & Steitz, J. A. (1974) Biochemistry 13, 21232129. 8. Kondo, M., Eggerston, G., Eisenstadt, J. & Lengyel, P. (1968) Nature (London) 220,368-370. 9. Cohen, S. S. & Lichtenstein, J. (1960) J. Biol. Chem. 235, 2112-2116. 10. Guarino, L. A. & Cohen, S. S. (1979) Anal. Biochem. 95, 7376. 11. Scafati, A. R., Stornaivolo, M. R. & Novaro, P. (1971) Biophys.
J. 11,370-374.
12. Hill, W. E., Rossetti, G. P. & VanHolde, K. E. (1969) J. Mol. Biol. 44, 263-277. 13. Weiss, R. L. & Morris, D. R. (1970) Biochim. Biophys. Acta 204, 502-511. 14. Weiss, R. L. & Morris, D. R. (1973) Biochemistry 12, 435441. 15. Kimes, B. W. & Morris, D. R. (1973) Biochemistry 12, 442449.
Vol. 76, No. 8, pp. 3660-3664, August 1979 Biochemistry
Mechanism of toxicity of putrescine in Anacystis nidulans (polyamines/cyanobacteria/spermidine/ribosomes)
LINDA A. GUARINO* AND SEYMOUR S. COHEN Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794
Contributed by Seymour S. Cohen, May 7, 1979
Putrescine is lethal to the cyanobacterium ABSTRACT Anacystis nidulans at extracellular pH values at which significant concentrations of the nonprotonated diamine rapidly diffuse into the cell and accumulate as the charged form. Although over 98% of the accumulated putrescine is not metabolized, a small fraction is rendered trichloroacetic acid-insoluble, and about 90% of this is bound as putrescine to proteins and cell structures. Various synthetic functions were studied in the presence of a bacteriostatic (40 pM) and a bacteriocidal (150 pM) concentration of putrescine at pH 9.5. Under lethal conditions, protein synthesis was completely inhibited after 45 min and CO2 fixation after 100 min, whereas nucleic acid synthesis was less affected. Spermidine was lost from the cell and its synthesis was arrested. These functions were much less inhibited at 40 pM putrescine. Ribosomes from putrescine-killed cells were found to be irreversibly dissociated into 30S and 50S subunits. Some putrescine (1-4 molecules) cosedimented with each subunit.
Putrescine is quite inhibitory to growth of the cyanobacterium Anacystis nidulans (1). The exogenous metabolite is toxic at the concentration (0.15 mM) at which putrescine is normally present within the organism. It appears that uncharged molecules of putrescine diffuse across the cell membrane and are protonated and trapped inside the cells (2). The pH gradient that exists across the cell membrane of these organisms growing under alkaline conditions permits a 500- to 3000-fold concentration of putrescine within the cells (2). The rapid uptake of putrescine to very high intracellular concentrations results in cell death. This paper presents data on the mechanism of this lethal effect. MATERIALS AND METHODS
Uptake of Putrescine and Viability of A. nidulans. A. nidulans strain 625 was grown as described (1). Exponential phase cultures were harvested by centrifugation at 10,000 X g for 5 min, washed, and resuspended at 1 X 108 cells per ml in Allen's medium containing 30 mM NaHCO3 and adjusted to the indicated pH values with NaOH. Cell number was determined in a Petroff-Hauser microscopic counting chamber. Uptake was initiated by the addition of [3H]putrescine at the indicated concentrations at specific activities of 16 or 62.5 ,tCi/,umol (1 Ci = 3.7 X 1010 becquerels). At various times aliquots were collected on Millipore HA filters and washed with water as described (2). Viability was determined by the single cell plating technique (3). Polyamine Assay. The procedures for dansylation of cell
extracts and fluorometric estimation of the dansyl polyamines after separation by thin-layer chromatography have been de-
scribed (1). Radioisotopes and Chemicals. [2,3-3H]Putrescine (20.64 Ci/mmol), [1,4-14C]putrescine (89.9 mCi/mmol), [tetramethylene-1,4-14C]spermidine (71.1 mCi/mmol), [2-14C]uracil The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
(45 mCi/mmol), [8-'4C]deoxyadenosine (38.8 mCi/mmol), [U-14C]leucine (45 mCi/mmol), sodium [14C]bicarbonate (9.3 mCi/mmol), and Liquifluor were purchased from New England Nuclear. Putrescine dihydrochloride, spermidine trihydrochloride, dansyl chloride, and proline were purchased from Calbiochem. Quantagram LQ6D thin-layer chromatography plates were obtained from Kontes. Synthesis of Macromolecules and CO2 Fixation. Cultures in exponential growth were harvested, washed, and resuspended at 8 X 107 cells per ml in Allen's medium (pH 9.5). DNA synthesis was assayed by the incorporation of [8-14C]deoxyadenosine (0.325 Atg/ml) (4). RNA synthesis was followed by the incorporation of [2-14C]uracil (0.5 Ag/ml) (5). [14C]Leucine was used to measure protein synthesis. Experiments were initiated by the addition of the appropriate isotope to separate flasks containing 20-ml cultures. At various times 0.5-ml aliquots were removed and added to an equal volume of cold 10% trichloroacetic acid. After 30 min on ice, the samples were filtered onto Whatman GF/C glass fiber filters and washed three times with 5 ml of cold 5% trichloroacetic acid and 5 ml of cold 95% (vol/vol) ethanol. The filters were dried and radioactivities were measured in Liquifluor. To determine the fraction of [2-14C]uracil incorporated into DNA and [814C]deoxyadenosine incorporated into RNA, the acid precipitates were incubated in 0.3 M KOH at 370C for 18 hr, cooled, precipitated with 0.5 M trichloroacetic acid (6), washed with 5% trichloroacetic acid onto filters, dried, and assayed for radioactivity. The RNA and DNA values given in the text include this correction. Free fatty acids and chlorophyll a were removed by extraction with cold acetone/methanol (7:2, vol/vol) and lipids were extracted by boiling in alcohol/ether (1:1). The lipid-free residue was incubated in 0.3 M KOH at 37°C for 18 hr to hydrolyze RNA. After precipitation with 0.6 M trichloroacetic acid the solution was centrifuged to remove the ribonucleotides and the pellet was heated in 0.3 M trichloroacetic acid at 80°C for 15 min. The solution was chilled and centrifuged to separate the protein fraction from the hydrolyzed DNA. Uptake and aminoacylation of ['4C]leucine were analyzed in growing cultures of A. nidulans in the presence of [14C]leucine at 0.5 ,uCi/ml. At 20 min and 60 min after the addition of leucine, 1-ml aliquots were removed and washed twice in fresh growth media. The cell pellet was extracted twice with cold 5% trichloroacetic acid. The cold-acid residue was extracted twice with hot 5% trichloroacetic acid (900C, 15 min) to hydrolyze [14C]leucyl-tRNA and extracts were pooled and assayed for radioactivity. CO2 fixation was measured as described (2). Isolation of Ribosomes. Ribosomes were isolated from 25-ml cultures by modifications of the method of Goldberg and Steitz (7). The cells were collected by centrifugation, washed twice in buffer A [100 mM NH4C1/10 mM Mg(OAc)2/20 mM Tris*
3660
Present address: Biophysics Laboratory, University of Wisconsin, Madison, WI 53706.
Biochemistry:
Guarino and Cohen
Proc. Natl. Acad. Sci. USA 76 (1979)
3661
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FIG. 1. Polyamine analyses of putrescine-treated cells. (A) Uptake of 150 MM [3H]putrescine (13.3 IACi/pumol) (A) at pH 9.5 and putrescine recovered from the cells and analyzed as dansyl putrescine (0). (B) Intracellular concentration of spermidine in presence (A) and absence (0) of 150 ,M putrescine. (C) Appearance of trichloroacetic acid-precipitable putrescine within the cells.
HCl (pH 7.5)/0.5 mM EDTA/3 mM mercaptoethanoll, and broken by sonication in buffer A. The resulting suspension was sedimented to remove cell walls and membranes at 37,000 X g, 40C, in the Sorvall SS34 rotor for 30 min; 3.5 ml of the resulting supernatant was layered over 5.0 ml of 1.1 M sucrose in buffer A-1 [50 mM NH4Cl/10 mM Mg(OAc)2/20 mM Tris-HCl (pH 7.5)/0.5 mM EDTA/3 mM mercaptoethanoll and centrifuged for 16 hr, 40C, at 48,000 rpm in a Beckman R 65 rotor. The ribosome pellet was gently resuspended in 0.5 ml of cold buffer A-1. Aliquots (200 jil) were layered over 5 ml of 5-20% sucrose gradients in buffer A-1 and centrifuged in a Beckman SW 50 rotor for 2 hr at 40,000 rpm, 40C (8). The gradients were collected from the top with an ISCO density gradient fractionator model 183, and the absorbance at 254 nm was monitored in an ISCO Model UA-4 absorbance monitor and simultaneously plotted on a Varian A-25 recorder. Ribosomal subunits were isolated in the same manner except that buffer A and A-1 contained 1 mM Mg(OAc)2. The ribosomal subunits were reassociated by adjusting the resuspended ribosomal subunits to 5 mM Mg(OAc)2 and 5 mM spermidine (9) and centrifuging in 5-20% sucrose gradients in buffer A-1 containing 5 mM Mg(OAc)2 and 5 mM spermidine.
RESULTS Polyamine Metabolism. To determine whether putrescine itself is the toxic agent in A. nidulans or whether it must first be metabolized to a toxic compound, Anacystis was incubated in the presence of [3H]putrescine for 14 hr. The amount of radioactively labeled putrescine that had been accumulated by
the cells was monitored by filtration and compared to the amount of putrescine that could be extracted from the cells and
quantitated after dansylation. As shown in Fig. 1A, the concentration of putrescine determined as dansyl putrescine was almost identical with the accumulation of radioactivity, indicating that there was very little metabolism of putrescine. Intracellular putrescine and internal radioactivity were maximal from 2 hr to 4 hr after the addition of putrescine and were then lost from the cells and appeared in the media at approximately the same rate. After 6 hr, total recoverable putrescine decreased gradually, indicating that putrescine was slowly degraded. However, approximately 0.2% of the 3H radioactivity in the cells was recovered in the trichloroacetic acid-insoluble residue
(Fig. 1C). The amount of trichloroacetic acid-precipitable radioactivity increased with time, and 90% of it was found in a lipid-free, nucleic acid-free protein fraction. After acid hydrolysis, the radioactive component was released as dansylatable putrescine. It could be calculated that at 1 hr there were approximately 70,000 trichloroacetic acid-precipitable putrescine molecules per cell. To examine the effect of putrescine on the internal concentration and synthesis of spermidine, a culture of A. nidulans was labeled with 1.4 MiM [14C]spermidine prior to exposure to 150 1AM [3H]putrescine. In the control cells, the concentration of spermidine increased 0.15 nmol/ml per hr for 14 hr (Fig. 1B) and the specific radioactivity of spermidine decreased with a half-life equal to the generation time (10). Under conditions of putrescine toxicity, spermidine increased 0.10 nmol/ml in the first hour, remained constant for another hour, and then was
Biochemistry: Guarino and Cohen
3662
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FIG. 2. Effects of putrescine on viability (B), CO2 fixation (C), and synthesis of protein (D), DNA (E), and RNA (F) in A. nidulans. Exponential phase cells were washed and resuspended in Allen's medium, pH 9.5, in 0 uM (-), 40 ,uM (-), or 150 ,uM (&) [3H]putrescine (16 ,Ci/,mol). The uptake of putrescine into whole cells is shown in A.
Biochemistry:
Proc. Nati. Acad. Sci. USA 76 (1979)
Guarino and Cohen
3663
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Irreversible FIG. 3. dissociation of ribosomes. Exponential phase cells were washed and resuspended in growth medium, pH 9.5, and incubated at 300C in the presence (-) or absence (---) of 150.uM putrescine for 2 hr. Ribosomes were isolated as described in Materials and Methods. (A) UV scan of ribosomes isolated and centrifuged in buffer A-1 containing 10 mM Mg24f (B) Ribosomes isolated and centrifuged in 1 mM Mg2+. (C) Ribosomes isolated in 1
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lost from the cells. The specific radioactivity of spermidine did not decrease after the first hour of incubation in the presence of putrescine, indicating that spermidine synthesis was inhibited. At 40,M putrescine, a nonlethal concentration, spermidine was not lost from the cells. Effect of Putrescine on the Synthesis of Macromolecules. Various synthetic activities, CO2 fixation, and DNA, RNA, and protein synthesis were monitored at two external concentrations of putrescine (40 and 150 MM) and in the absence of external putrescine at pH 9.5 (Fig. 2). The rates of putrescine-accumulation are shown in Fig. 2A. In the 40 MM culture the uptake of putrescine was linear for 120 min, at which time the intracellular concentration was 35 mM. As shown in Fig. 2B, this had a bacteriostatic effect. In the culture treated with 150 ,M putrescine, uptake was linear for 100 min and leveled off at an intracellular concentration equal to 87 mM. After 180 min, the intracellular putrescine slowly began to leak out of the cells. In this culture (Fig. 2B) the cells rapidly lost viability, leaving approximately 700 viable cells per ml. Of the four synthetic processes analyzed, protein synthesis was the first inhibited by putrescine. In the culture incubated in the presence of 150 MM putrescine, protein synthesis was abruptly arrested 35-40 min after addition of the diamine (Fig. 2D). In the 40MM-treated culture, the rate of protein synthesis was decreased 15% compared to the control. The inhibition of protein synthesis was probably not due to an inhibition of uptake or aminoacylation of [14C]leucine, as determined by
30 35 25 20 15 Fraction FIG. 4. The cosedimentation of trichloroacetic acid-precipitable putrescine and ribosomes. Ribosomes were isolated as described in the legend to Fig. 3A. Two-drop fractions were collected onto filters and precipitated in 5% trichloroacetic acid ( --- ). -, UV tracing.
5
10
analysis of intracellular [14C]leucine in cold and hot 5% trichloroacetic acid extracts, respectively. The levels of radioactivity in these fractions were equal to those in the controls. CO2 fixation (Fig. 2C) was not appreciably affected by the presence of 40,uM putrescine throughout the experiment. In the culture treated with 150 MM putrescine, CO2 was fixed at the control rate for 100 min and then the rate of CO2 fixation was decreased to 4% of the control rate. The synthesis of RNA (Fig. 2F) was not affected by 40 MM putrescine, whereas it was inhibited slightly 30 min after the addition of 150 MuM putrescine and slowed down more rapidly after 100 min. The incorporation of [14C]deoxyadenosine appears to be initially increased by the addition of 40 M putrescine (Fig. 2E). This could be due to an effect of putrescine on the permeability of deoxyadenosine, which normally enters the cells very slowly (4). DNA was synthesized in the 150 MuM-culture at a constant rate, which was about 60% of the control rate. Because protein synthesis was inhibited first, the effect of putrescine accumulation on the integrity of the protein synthetic machinery was examined further. Effect of Putrescine on Ribosome Structure. Cells were incubated in the presence or absence of 150,uM putrescine at pH 9.5 for 2 hr. Ribosomes were isolated in the presence of 10 mM Mg2+ and analyzed in sucrose gradients (Fig. 3A). In the control cells, 85% of the ribosomes were present as 70S ribosomes, whereas in the putrescine-treated culture, 96% of the ribosomes were dissociated into 30S and 50S subunits. Ribosomes from control cells isolated in 1. mM Mg2+ dissociated into
Biochemistry: Guarino and Cohen
3664
IntraExternal cellular putrescine, putrescine, mM* AuM 0 1
40 100 150
0.15 1.2 15.3 68.4 100.1
Proc. Nati. Acad. Sci. USA 76 (1979)
Table 1. Summary of the metabolic effects of putrescine Molecules putrescine per cell Rate of proRibosome Cell walls tein synthesist dissociation,§ + Ribosomal Soluble Lethalityt % inhibition membranes % pellet proteins 1.14
0
9
NA
NA
1.14 1.00 0.4 0.03
0 10 88 96
13.5 23.5 58.0 78.0
85,000 1,359,000 915,000
3,000 46,000 15,000 20,000
891,000
NA 4,000 35,000 68,000
91,000
Molecules putrescine per ribosomal unit 30S 50S 70S NA
NA
NA
0.3 1.5 0.5 0.5
0.3 1.6 0.5 1.0
0.1 0.7 1.1 1.9
NA, not applicable. * Intracellular concentration of putrescine at equilibrium 100-150 min after addition of putrescine. t Lethality expressed as viable count at 120 min/viable count at zero time. Calculated 120 min after addition of putrescine. § Percent of total absorbance present as 30S and 50S subunits, 120 min after addition of putrescine; average of duplicate determinations.
50S and 30S subunits (Fig. 3B), which then readily reassociated into 70S ribosomes in the presence of 5 mM Mg2+ and 5 mM spermidine (Fig. 3C). Ribosomes isolated from putrescinetreated cells in 1 mM Mg2+ were dissociated into several small components (Fig. 3B). In the presence of 5 mM Mg2+ and 5 mM spermidine, they reformed 30S and 50S subunits with a small peak at 70S. The addition of 50 mM putrescine to purified 70S control ribosomes in vitro did not dissociate the ribosomes. A possible relationship between trichloroacetic acid-insoluble putrescine and ribosome dissociation was then examined. Ribosomes were isolated, as described above, from cells that had been incubated in the presence of 150 ,uM [14C]putrescine for 2 hr. As shown in Fig. 4, radioactivity was associated with 30S and 50S subunits. The ribosomes were hydrolyzed with acid (6 M HC1, 110°C, 18 hr) and dansylated. The radioactive dansyl derivative isolated from the ribosomes comigrated with standard dansylputrescine, indicating that putrescine was not metabolized prior to binding to the ribosomes. On the basis of input specific radioactivity (26.6 ,uCi/,umol), extinction coefficients (11), and molecular weights (12) of ribosomes and subunits, there were 2.7 molecules of putrescine per 30S subunit and 4.3 molecules of putrescine per 50S subunit. In a separate experiment, the peak fractions were collected and resedimented through sucrose gradients. The radioactivity due to putrescine comigrated with the ribosome peaks. in the second gradient. In this experiment, there were 1.6 and 3.4 molecules of putrescine per 30S and 50S
subunit, respectively. Summary of the Metabolic Effects of Putrescine. Cultures of A. nidulans were incubated in the presence of the indicated concentrations of [14C]putrescine. Uptake and accumulation of putrescine were monitored continuously for 3 hr. After 2 hr, 25 ml was removed from each culture and ribosomes were isolated. In Table 1, it can be seen that the lethal action of putrescine correlates fairly well with an inhibition of protein synthesis and the dissociation of 70S ribosomes. The distribution of radioactive putrescine recovered in the various cell fractions and in the ribosomal subunits is shown in the last columns. In these columns it would appear that the extent of covalent binding of putrescine to the cell fractions at 2 hr is decreased by cell killing. A clear relation of acid-insoluble ribosomal putrescine to the dissociation of the ribosomes has not been demonstrated. DISCUSSION The addition of toxic concentrations of putrescine to growing cultures of A. nidulans results in a disruption of several metabolic functions within the cells. Complete inhibition of protein synthesis was the earliest event observed after addition of 150
,uM putrescine to the growth medium. This inhibition of protein synthesis was accompanied by an irreversible dissociation of ribosomes. The concentrations of various ions are very important in maintaining intact, functional, 70S ribosomes. In vitro dialysis of purified Escherichia coli 70S ribosomes against spermidine or putrescine can lead to-a stoichiometric replacement of Mg2+ with polyamine, resulting in dissociation (13), irreversible conformational alterations, loss of polymerizing activity, and loss of specific proteins from the 50S subunit (14, 15). Spermidine has also been found to contribute to ribosome stability (9). When A. nidulans was incubated in 150 ,uM putrescine, cellular spermidine was lost from the cell. It is probable that some of the spermidine originally bound to the ribosomes was replaced by putrescine. Putrescine at the high intracellular concentrations observed under conditions of lethality and ribosomal dissociation could compete for various binding sites on the ribosomes, thereby displacing other cations and leading to dissociation. It is possible also that the displacement of essential cations from the ribosomes may also open certain specific sites for covalent binding by putrescine; this possibility of specific inactivation of the ribosomal subunits has yet to be tested. We thank Drs. Dennis Hruby and Richard Condit for helpful discussions. This work was supported by Grant PCM 78-04324 from the National Science Foundation. 1. Ramakrishna, S., Guarino, L. A. & Cohen, S. S. (1978) J. Bacteriol. 134,744-750. 2. Guarino, L. A. & Cohen, S. S. (1979) Proc. Natl. Acad. Sci. USA
76,3184-3188.
3. Allen, M. M. (1968) J. Phycol. 4, 1-4. 4. Restaino, L. & Frampton, E. W. (1975) J. Bacteriol. 124, 155160. 5. Pigott, G. H. & Carr, N. G. (1971) Arch. Microbiol. 79, 1-6. 6. Stern, J. L., Sekiguchi, M., Barner, H. D. & Cohen, S. S. (1964) J. Mol. Biol. 8,629-637. 7. Goldberg, M. L. & Steitz, J. A. (1974) Biochemistry 13, 21232129. 8. Kondo, M., Eggerston, G., Eisenstadt, J. & Lengyel, P. (1968) Nature (London) 220,368-370. 9. Cohen, S. S. & Lichtenstein, J. (1960) J. Biol. Chem. 235, 2112-2116. 10. Guarino, L. A. & Cohen, S. S. (1979) Anal. Biochem. 95, 7376. 11. Scafati, A. R., Stornaivolo, M. R. & Novaro, P. (1971) Biophys.
J. 11,370-374.
12. Hill, W. E., Rossetti, G. P. & VanHolde, K. E. (1969) J. Mol. Biol. 44, 263-277. 13. Weiss, R. L. & Morris, D. R. (1970) Biochim. Biophys. Acta 204, 502-511. 14. Weiss, R. L. & Morris, D. R. (1973) Biochemistry 12, 435441. 15. Kimes, B. W. & Morris, D. R. (1973) Biochemistry 12, 442449.
