ANALYTICAL
BIOCHEMISTRY
The Estimation
95, 73-76 (1979)
of Turnover LINDA
Department
of Pharmacological
of Spermidine
in Anacystis
A. GUARINO
AND SEYMOUR
S. COHEN
Sciences,
University
York.
State
of Nell,
Stony
Brook,
nidulans’
Nelc, York
I1794
Received October 8. 1978 The turnover of spermidine in Anacystis nidulans was studied using [2-Ylmethionine to prelabel intracellular spermidine. It was found that there is essentially neither excretion nor degradation of spermidine in exponentially gr0wingAnacysti.s nidukms. Spermidine was degraded rapidly in stationary phase cells. The half-life of specific activity of spermidine in exponential phase was 8.3 h, a period similar to that of the doubling time (7.5 h) of the bacterium. The rate of synthesis of spermidine was calculated to be 0.04 nmol/lOn cells/h.
Polyamines have been implicated as regulators of cellular functions due to their increased synthesis under conditions in which cell growth is stimulated. In particular, ornithine decarboxylase (ODC),* a rate-limiting enzyme for polyamine biosynthesis in the mammal, has been shown to rapidly increase in activity in response to a variety of factors, as summarized in several reviews (l-3). A regulatory role for cellular polyamines would suggest that the polyamine levels should increase significantly and return to normal after the growth stimulus has been removed. Although very little work has been done on the metabolism and turnover of polyamines, McCormick (4) has shown that in mouse fibroblasts, the turnover rates of spermidine and spermine were very low in exponential phase cells, indicating that rapid fluctuations in total cell polyamine were unlikely to occur. Nevertheless this result did not exclude localized short-term changes within cellular compartments. In Escherichia coli, polyamines have also been implicated in important regulatory roles. In particular, there is a very strong ’ This paper is dedicated to the memory of Dr. Alvin Nason. 2 Abbreviations used: ODC. ornithine decarboxylase; SPD. spermidine; SPD*. radioactive spermidine; PCA, perchloric acid.
correlation between RNA synthesis and spermidine synthesis and metabolism (1,5). Tabor and Dobbs (6) have shown that there is retention of prelabeled spermidine in the exponential phase although the polyamines are metabolized to acetyl derivatives when harvested at low temperature. In stationary phase E. co/i (7), spermidine is found as a glutathionyl derivative. In a study from this laboratory (5), the increase of intracellular spermidine during biosynthesis of polyamine in the absence of growth was minimized by acetylation of spermidine and excretion of acetyl spermidine into the medium. In a procaryotic photoautotroph, Amcystis nidulans, spermidine metabolism was found to be significantly different from that of E. co/i (8). Stationary phase cells do not synthesize or excrete spermidine conjugates and appear to degrade spermidine completely. When spermidine is added to exponentially growing cells, intracellular spermidine concentration increases greatly and is then rapidly degraded into diaminopropane. It was of interest to determine whether this turnover of spermidine occurred during exponential growth with normally synthesized spermidine, which is present at a total concentration of 1 to 2 mM. The kinetic analysis of McCormick (4), developed for animal cells in tissue culture, was then used 73
0003-2697/79/070073-04$02.00/O CopyrIght All r,aht\
I 1979 by Academtc E’rr\r. inc. of ,rproducl,,,n in ;tnv form ,c\encd
74
GUARINO
to determine whether endogenous spermidine was degraded in exponentially growing Anacystis nidulans.
Growth of Anacystis nidulans. Strain 625 from the Indiana Culture Collections was grown at 30°C with rotary shaking in Allen’s medium containing 1% NaHCO, with illumination from the top at 7 to 8 W/cm2, as previously described (8). Growth was monitored by measuring the increase in turbidity in a Klett calorimeter (66 filter). Cell number was determined in the Petroff-Hauser microscopic counting chamber. Polyamine estimation. Intracellular spermidine was labeled by growing cells in the presence of 0.25 &i/ml of DL-[2-‘*Clmethionine (5.78 mCi/mmol, New England Nuclear, Boston, Mass.) for 12 h. Cells were then washed three times with fresh growth media to remove exogenous radioactivity and diluted to approximately 8 x 10’ cells/ml. At the indicated times, 2- to S-ml samples were removed, centrifuged and the cells were extracted in 0.5 ml cold 5% perchloric acid. The procedures for dansylation of cell extracts and fluorometric estimation of the dansyl polyamines after separation by thin-layer chromatography (tic) have been described (8). The radioactivity in spermidine was determined by scraping dansyl spots from the tic plates and counting in 3a70 scintillation fluid (Research Products International; Elk Grove Village, Ill). Interpretation of the data. The equations derived by McCormick (4) were used to analyze the results. Briefly, the equations which apply are as follows. Rate of decrease of radioactive (SPD) = -k,JSPD*],
one-half is (ln2)/k,. When cells are in the exponential phase of growth, total spermidine accumulates as follows: rate of accumulation
METHODS
spermidine
AND COHEN
[l]
where k, is the rate constant for decrease of the radioactivity and [SPD*] is the total amount of radioactive spermidine in cells per unit volume. The time required to decrease the radioactivity in spermidine by
of SPD
= k,WDl,
[21
where k, is a rate constant for accumulation of spermidine and [SPD] is the amount of spermidine in cells per unit volume of culture. The time required to double the amount of spermidine in cells per unit volume is (ln2)/k,. The specific radioactivity of spermidine, [SPD*]/[SPD], is obtained by dividing the above equations and the time required to decrease the specific radioactivity by one-half (tlhz,,) is: t lhsa = (ln2)/( kd + k,).
[31 Since nonradioactive SPD is part of the same pool as radioactive SPD, Eq. [l] holds for total SPD in the cell and the rate of accumulation is actually a function of the rate of synthesis and the rate of loss of SPD or: rate of synthesis of SPD = (ln2) * [SPD]It&
[4]
If the amount of spermidine per cell (a) is constant during exponential phase, then: rate of synthesis of SPD per cell = (ln2)+crltY2,,.
ES]
RESULTS Growth and Accumulation
of Spermidine
When exponential phase cells were washed and resuspended in fresh growth media, the culture continued to increase exponentially without an appreciable lag phase (Fig. 1A). The culture continued to grow logarithmically for 49 h. At high cell densities, light scattering and self-absorption interfere with the Klett readings, leading to an underestimation of cell growth at high turbidities. The increase in intracellular spermidine was exponential for 27 h; during this time interval, the amount of spermidine per
ESTIMATION
OF SPERMIDINE
TURNOVER
cell was constant and equal to 0.48 nmoY108 cells. In late log phase (27-49 h), cellular spermidine increased only slightly (1 nmoYm1). Increase in cell turbidity leveled off between 49 and 60 h; during this time interval, the concentration of intracellular spermidine rapidly decreased. Spermidine could not be recovered in the medium and appears to be degraded. This phenomenon has been described earlier (8). Turnover of Spermidine
After 12 h of prelabeling with DL-[~-'~C]methionine, 65% of the radioactivity in the cells was recovered in the PCA-insoluble fraction and 35% in the PCA-soluble fraction. Spermidine accounted for 55% of the PCA-soluble radioactivity; the remainder of the soluble radioactivity was not extracted into benzene during the dansylation procedure. Since the isotope was added as both A.
3.
-u
HOURS
FIG. 1. Growth of Anacysris nidulans and accumulation of spermidine. Growth was estimated by monitoring the increase in turbidity in a Klett calorimeter (A). At the indicated times, samples were removed for quantitation of spermidine (B) as described under Methods.
FIG. 2. Turnover and decrease in specific radioactivity of cellular spermidine. Exponential phase cells were grown in the presence of [2-Wjmethionine for 12 h after washing to remove exogenous methionine and the cells were resuspended in fresh growth media lacking methionine. The radioactivity present in cellular spermidine (0) and the specific radioactivity of cellular spermidine (A.) were determined as described under Methods.
the D- and L-isomers, it could be expected that the o-isomer would not be converted to S-adenosylmethionine and that D-methionine might accumulate in the cells. After removal of exogenous methionine. by centrifugation and washing, the amount of radioactivity present in spermidine remains constant for 49 h as shown in Fig. 2. This indicates that there is essentially no metabolism of spermidine in exponential or early stationary phase cells. After cells reached stationary phase, radioactivity was rapidly lost from the cells. All of this radioactivity was recovered as acid-soluble material in the medium. Analysis of the medium by the dansyl procedure showed that the radioactivity was not present in spermidine, as described earlier (8). In this experiment there is no loss of ra-
76
GUARINO
AND COHEN
implies that there must be some control on the enzyme which degrades spermidine durof 0.30 h-’ and the half-life of decrease is ing stationary phase and during exponential about 2.3 h. phase when spermidine is added to the The specific activity of spermidine shown growth media (8). The control mechanism of in Fig. 2 can be obtained by dividing the this enzyme is interesting, since it has been counts per unit volume by the total suggested that Anacystis niduhs is incaspermidine present per unit volume. As pable of controlling metabolism through ingiven earlier, the time required to decrease duction or repression of enzyme synthesis (9,lO). However, one example of induction the specific radioactivity by one-half (t&J has been demonstrated recently (11). is (ln2)/(k, + k,) (Eq. [3]). In exponential phase, the decrease in specific radioactivity The relatively high specific radioactivity in cell spermidine is only a function of de (6.3 cpm/pmol) of spermidine obtained after the 12 h prelabel from DL-[2-14C]methionine nova synthesis of unlabeled spermidine. The time required to double total cellular (12.7 cpm/pmol) suggests strongly that mespermidine or to decrease its specific thionine is a precursor of spermidine. This activity to one-half was 8.3 h. This is close appears to eliminate a major role of the to the doubling time of the cells in this newly described Tait pathway (12) in which the aminopropyl moiety is derived from experiment (7.5 h). aspartate. dioactivity
during exponential
phase, i.e.,
k, = 0. In stationary phase, kd is of the order
Rate of Synthesis of Spermidine
The rate of synthesis of spermidine can be calculated from its amount per cell and the half-life of its specific radioactivity. The concentration of spermidine in exponential phase cells was 0.48 nmol/108 cells (Fig. 1). The half-life of its specific radioactivity (Fig. 2) was found to be 8.3 h. From Eq. [5] we find that the rate of synthesis per lo8 cells is 0.040 nmollh. DISCUSSION
The method of analysis described by McCormick for polyamines in animal cell cultures appears entirely applicable to this procaryotic system. Indeed it is an even simpler system than that of HeLa or L cells which convert spermidine to spermine. In Anacystis which lacks spermine, there is no significant transformation of spermidine during exponential growth. In this sense spermidine in Anacystis is behaving like spermine in the animal cells. The lack of turnover of spermidine during exponential growth of Anacystis nidulans
ACKNOWLEDGMENT This work was supported by Grant PCM78-04324 from the National Science Foundation.
REFERENCES 1. Cohen, S. S. (1971) Introduction to the Polyamines, Prentice-Hall, Englewood Cliffs, N. J. Bachrach, U. (1973) Function of Naturally Occurring Polyamines, Academic Press, New York. JPnne, J., P&6, H., and Raina, A. (1978)Biochem. Riophys. Acta 413, 241-293. McCormick, F. (1978) Biochem J. 174,427-434. Cohen, S. S., Hoffner, N., Jensen, M., Moore, M., and Raina, A. (1967) Proc. Nat. Acad. Sri. 57, 721-728. 6. Tabor, C. W., and Dobbs, L. G. (1970) J. Biol. Chem. 245, 2086-2091. 7. Tabor, H., and Tabor, C. W. (1976) fral. J. Biothem.
25,70-76.
8. Ramakrishna, S., Guarino, L., and Cohen, S. S. (1978) J. Bacterial. 134, 744-750. 9. Delaney, S. F., Dickson, A., and Carr, N. G. (1973) J. Gen.
Microbial.
79, 89-94.
10. Mann, N., and Carr, N. G. (1974) J. Gen. Microbiol.
83, 399-405.
11. Singer, R. A., and Doolittle, W. F. (197.5) Nature (London) 253, 650-651. 12. Tait, G. H. (1976)Biochem. Sot. Trans. 4,610-612.
BIOCHEMISTRY
The Estimation
95, 73-76 (1979)
of Turnover LINDA
Department
of Pharmacological
of Spermidine
in Anacystis
A. GUARINO
AND SEYMOUR
S. COHEN
Sciences,
University
York.
State
of Nell,
Stony
Brook,
nidulans’
Nelc, York
I1794
Received October 8. 1978 The turnover of spermidine in Anacystis nidulans was studied using [2-Ylmethionine to prelabel intracellular spermidine. It was found that there is essentially neither excretion nor degradation of spermidine in exponentially gr0wingAnacysti.s nidukms. Spermidine was degraded rapidly in stationary phase cells. The half-life of specific activity of spermidine in exponential phase was 8.3 h, a period similar to that of the doubling time (7.5 h) of the bacterium. The rate of synthesis of spermidine was calculated to be 0.04 nmol/lOn cells/h.
Polyamines have been implicated as regulators of cellular functions due to their increased synthesis under conditions in which cell growth is stimulated. In particular, ornithine decarboxylase (ODC),* a rate-limiting enzyme for polyamine biosynthesis in the mammal, has been shown to rapidly increase in activity in response to a variety of factors, as summarized in several reviews (l-3). A regulatory role for cellular polyamines would suggest that the polyamine levels should increase significantly and return to normal after the growth stimulus has been removed. Although very little work has been done on the metabolism and turnover of polyamines, McCormick (4) has shown that in mouse fibroblasts, the turnover rates of spermidine and spermine were very low in exponential phase cells, indicating that rapid fluctuations in total cell polyamine were unlikely to occur. Nevertheless this result did not exclude localized short-term changes within cellular compartments. In Escherichia coli, polyamines have also been implicated in important regulatory roles. In particular, there is a very strong ’ This paper is dedicated to the memory of Dr. Alvin Nason. 2 Abbreviations used: ODC. ornithine decarboxylase; SPD. spermidine; SPD*. radioactive spermidine; PCA, perchloric acid.
correlation between RNA synthesis and spermidine synthesis and metabolism (1,5). Tabor and Dobbs (6) have shown that there is retention of prelabeled spermidine in the exponential phase although the polyamines are metabolized to acetyl derivatives when harvested at low temperature. In stationary phase E. co/i (7), spermidine is found as a glutathionyl derivative. In a study from this laboratory (5), the increase of intracellular spermidine during biosynthesis of polyamine in the absence of growth was minimized by acetylation of spermidine and excretion of acetyl spermidine into the medium. In a procaryotic photoautotroph, Amcystis nidulans, spermidine metabolism was found to be significantly different from that of E. co/i (8). Stationary phase cells do not synthesize or excrete spermidine conjugates and appear to degrade spermidine completely. When spermidine is added to exponentially growing cells, intracellular spermidine concentration increases greatly and is then rapidly degraded into diaminopropane. It was of interest to determine whether this turnover of spermidine occurred during exponential growth with normally synthesized spermidine, which is present at a total concentration of 1 to 2 mM. The kinetic analysis of McCormick (4), developed for animal cells in tissue culture, was then used 73
0003-2697/79/070073-04$02.00/O CopyrIght All r,aht\
I 1979 by Academtc E’rr\r. inc. of ,rproducl,,,n in ;tnv form ,c\encd
74
GUARINO
to determine whether endogenous spermidine was degraded in exponentially growing Anacystis nidulans.
Growth of Anacystis nidulans. Strain 625 from the Indiana Culture Collections was grown at 30°C with rotary shaking in Allen’s medium containing 1% NaHCO, with illumination from the top at 7 to 8 W/cm2, as previously described (8). Growth was monitored by measuring the increase in turbidity in a Klett calorimeter (66 filter). Cell number was determined in the Petroff-Hauser microscopic counting chamber. Polyamine estimation. Intracellular spermidine was labeled by growing cells in the presence of 0.25 &i/ml of DL-[2-‘*Clmethionine (5.78 mCi/mmol, New England Nuclear, Boston, Mass.) for 12 h. Cells were then washed three times with fresh growth media to remove exogenous radioactivity and diluted to approximately 8 x 10’ cells/ml. At the indicated times, 2- to S-ml samples were removed, centrifuged and the cells were extracted in 0.5 ml cold 5% perchloric acid. The procedures for dansylation of cell extracts and fluorometric estimation of the dansyl polyamines after separation by thin-layer chromatography (tic) have been described (8). The radioactivity in spermidine was determined by scraping dansyl spots from the tic plates and counting in 3a70 scintillation fluid (Research Products International; Elk Grove Village, Ill). Interpretation of the data. The equations derived by McCormick (4) were used to analyze the results. Briefly, the equations which apply are as follows. Rate of decrease of radioactive (SPD) = -k,JSPD*],
one-half is (ln2)/k,. When cells are in the exponential phase of growth, total spermidine accumulates as follows: rate of accumulation
METHODS
spermidine
AND COHEN
[l]
where k, is the rate constant for decrease of the radioactivity and [SPD*] is the total amount of radioactive spermidine in cells per unit volume. The time required to decrease the radioactivity in spermidine by
of SPD
= k,WDl,
[21
where k, is a rate constant for accumulation of spermidine and [SPD] is the amount of spermidine in cells per unit volume of culture. The time required to double the amount of spermidine in cells per unit volume is (ln2)/k,. The specific radioactivity of spermidine, [SPD*]/[SPD], is obtained by dividing the above equations and the time required to decrease the specific radioactivity by one-half (tlhz,,) is: t lhsa = (ln2)/( kd + k,).
[31 Since nonradioactive SPD is part of the same pool as radioactive SPD, Eq. [l] holds for total SPD in the cell and the rate of accumulation is actually a function of the rate of synthesis and the rate of loss of SPD or: rate of synthesis of SPD = (ln2) * [SPD]It&
[4]
If the amount of spermidine per cell (a) is constant during exponential phase, then: rate of synthesis of SPD per cell = (ln2)+crltY2,,.
ES]
RESULTS Growth and Accumulation
of Spermidine
When exponential phase cells were washed and resuspended in fresh growth media, the culture continued to increase exponentially without an appreciable lag phase (Fig. 1A). The culture continued to grow logarithmically for 49 h. At high cell densities, light scattering and self-absorption interfere with the Klett readings, leading to an underestimation of cell growth at high turbidities. The increase in intracellular spermidine was exponential for 27 h; during this time interval, the amount of spermidine per
ESTIMATION
OF SPERMIDINE
TURNOVER
cell was constant and equal to 0.48 nmoY108 cells. In late log phase (27-49 h), cellular spermidine increased only slightly (1 nmoYm1). Increase in cell turbidity leveled off between 49 and 60 h; during this time interval, the concentration of intracellular spermidine rapidly decreased. Spermidine could not be recovered in the medium and appears to be degraded. This phenomenon has been described earlier (8). Turnover of Spermidine
After 12 h of prelabeling with DL-[~-'~C]methionine, 65% of the radioactivity in the cells was recovered in the PCA-insoluble fraction and 35% in the PCA-soluble fraction. Spermidine accounted for 55% of the PCA-soluble radioactivity; the remainder of the soluble radioactivity was not extracted into benzene during the dansylation procedure. Since the isotope was added as both A.
3.
-u
HOURS
FIG. 1. Growth of Anacysris nidulans and accumulation of spermidine. Growth was estimated by monitoring the increase in turbidity in a Klett calorimeter (A). At the indicated times, samples were removed for quantitation of spermidine (B) as described under Methods.
FIG. 2. Turnover and decrease in specific radioactivity of cellular spermidine. Exponential phase cells were grown in the presence of [2-Wjmethionine for 12 h after washing to remove exogenous methionine and the cells were resuspended in fresh growth media lacking methionine. The radioactivity present in cellular spermidine (0) and the specific radioactivity of cellular spermidine (A.) were determined as described under Methods.
the D- and L-isomers, it could be expected that the o-isomer would not be converted to S-adenosylmethionine and that D-methionine might accumulate in the cells. After removal of exogenous methionine. by centrifugation and washing, the amount of radioactivity present in spermidine remains constant for 49 h as shown in Fig. 2. This indicates that there is essentially no metabolism of spermidine in exponential or early stationary phase cells. After cells reached stationary phase, radioactivity was rapidly lost from the cells. All of this radioactivity was recovered as acid-soluble material in the medium. Analysis of the medium by the dansyl procedure showed that the radioactivity was not present in spermidine, as described earlier (8). In this experiment there is no loss of ra-
76
GUARINO
AND COHEN
implies that there must be some control on the enzyme which degrades spermidine durof 0.30 h-’ and the half-life of decrease is ing stationary phase and during exponential about 2.3 h. phase when spermidine is added to the The specific activity of spermidine shown growth media (8). The control mechanism of in Fig. 2 can be obtained by dividing the this enzyme is interesting, since it has been counts per unit volume by the total suggested that Anacystis niduhs is incaspermidine present per unit volume. As pable of controlling metabolism through ingiven earlier, the time required to decrease duction or repression of enzyme synthesis (9,lO). However, one example of induction the specific radioactivity by one-half (t&J has been demonstrated recently (11). is (ln2)/(k, + k,) (Eq. [3]). In exponential phase, the decrease in specific radioactivity The relatively high specific radioactivity in cell spermidine is only a function of de (6.3 cpm/pmol) of spermidine obtained after the 12 h prelabel from DL-[2-14C]methionine nova synthesis of unlabeled spermidine. The time required to double total cellular (12.7 cpm/pmol) suggests strongly that mespermidine or to decrease its specific thionine is a precursor of spermidine. This activity to one-half was 8.3 h. This is close appears to eliminate a major role of the to the doubling time of the cells in this newly described Tait pathway (12) in which the aminopropyl moiety is derived from experiment (7.5 h). aspartate. dioactivity
during exponential
phase, i.e.,
k, = 0. In stationary phase, kd is of the order
Rate of Synthesis of Spermidine
The rate of synthesis of spermidine can be calculated from its amount per cell and the half-life of its specific radioactivity. The concentration of spermidine in exponential phase cells was 0.48 nmol/108 cells (Fig. 1). The half-life of its specific radioactivity (Fig. 2) was found to be 8.3 h. From Eq. [5] we find that the rate of synthesis per lo8 cells is 0.040 nmollh. DISCUSSION
The method of analysis described by McCormick for polyamines in animal cell cultures appears entirely applicable to this procaryotic system. Indeed it is an even simpler system than that of HeLa or L cells which convert spermidine to spermine. In Anacystis which lacks spermine, there is no significant transformation of spermidine during exponential growth. In this sense spermidine in Anacystis is behaving like spermine in the animal cells. The lack of turnover of spermidine during exponential growth of Anacystis nidulans
ACKNOWLEDGMENT This work was supported by Grant PCM78-04324 from the National Science Foundation.
REFERENCES 1. Cohen, S. S. (1971) Introduction to the Polyamines, Prentice-Hall, Englewood Cliffs, N. J. Bachrach, U. (1973) Function of Naturally Occurring Polyamines, Academic Press, New York. JPnne, J., P&6, H., and Raina, A. (1978)Biochem. Riophys. Acta 413, 241-293. McCormick, F. (1978) Biochem J. 174,427-434. Cohen, S. S., Hoffner, N., Jensen, M., Moore, M., and Raina, A. (1967) Proc. Nat. Acad. Sri. 57, 721-728. 6. Tabor, C. W., and Dobbs, L. G. (1970) J. Biol. Chem. 245, 2086-2091. 7. Tabor, H., and Tabor, C. W. (1976) fral. J. Biothem.
25,70-76.
8. Ramakrishna, S., Guarino, L., and Cohen, S. S. (1978) J. Bacterial. 134, 744-750. 9. Delaney, S. F., Dickson, A., and Carr, N. G. (1973) J. Gen.
Microbial.
79, 89-94.
10. Mann, N., and Carr, N. G. (1974) J. Gen. Microbiol.
83, 399-405.
11. Singer, R. A., and Doolittle, W. F. (197.5) Nature (London) 253, 650-651. 12. Tait, G. H. (1976)Biochem. Sot. Trans. 4,610-612.
