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The effects of steroids on the growth of Euglena gracilis
ARCHIVEG
OF
BIOCHEMISTRY
AND
BIOPHYSICS
73,
%ii-‘i!%
(1957)
LETTERS TO THE EDITORS The Effects of Steroids on the Growth of Euglena
gracitislJ
Steroid effects in mammalian systems have been well investigated, and indeed are sufficiently well understood so that a given program of events can be described in general terms when single steroids or combinations of steroids are introduced into the experimental system. Many steroid hormone effects are reflected in cell proliferation, or “growth,” while combinations of hormones show competition with each other aa reflected in similar criteria (1). The question arises whether it is possible to demonstrate a similar program of events as a result of the introduction of steroids into a suitable single cell system. Such a system must not only show a response to single steroids but should respond to multiple steroids in a pattern that would show some of the interrelationships that have already been demonstrated in fhe mammal. WhiIe admittedly the metazoan system and the single cell system may be very different, such a demonstration could be used to investigate any generalized function of steroids at the cellular level and might even be used to investigate structure requirements of steroids for hormone action. In this communication we wish to report investigations on a single cell system that appears to permit study, by means of growth measurements, of not only a response to characteristic steroid hormones, but also of the interaction or “competition” between combinations of such steroids. The single cell used was a streptomycin-bleached free-living flagellate, Euglena gracilis var. bacillaris. This chlorotic Euglena may be cultivated on a defined medium, does not require sterols or steroids for its growth and avoids the problems encountered with its photosynthetic counterpart. Testosterone, 17-hydroxycorticosterone (Compound F), and estrone were chosen as characteristic steroid hormones while cholesterol was used as a control compound that contains the characteristic steroid nucleus but has no known hormone activity. In these studies growth was measured as optical density at 570 rnp. Cultures were grown in 10 ml. of Cramer-Myers acetate medium (2) contained in 30 ml. screwcap tubes which were shaken in the dark throughout the period of culture growth. All steroid concentrations were within the water solubility range of each steroid (3). The crystalline steroid was added directly to the culture medium during its preparation. Inoculum of 0.1 ml. of organisms from a culture in the log phase of growth was used for each tube. Controls using no hormone, no carbon source and 1 From a dissertation submitted in partial fulfillment of the requirements for the degree of Master of Arts in the University of California, Los Angeles, 1956. * These studies were aided by a contract between the Office of Naval Research, Department of the Navy, and the University of California, Los Angeles, NR 120-336. 273
274
LETTERS
TO
THE
EDITORS
TABLE I Diflerent Concentrations of Steroids and Mean Generation Time- (WC., pH 6.8) GT (hours) C~peAt$$ Ill. I
0 (control) 0.1 0.2 0.5 1.0 1.5 2.0
Estrone
18.80 14.29 16.09 19.02 21.15 -
Testosterone
18.80 15.85 18.72 20.10 21.24
1f -HYdrOWcorticosterone
18.80 17.18 16.38 19.06 21.12
Cholesterol
18.80 18.41 19.03 18.30 -
0 Each dose-response curve was run at least twice and generally three times with triplicate samples each time. Statistical analysis by an analysis of variance shows that the dose-response correlation using the steroid hormones is significant at the 5aj, level in all cases. no hormone, and hormone but no carbon source were run with each experiment. In the latter two controls no growth was observed in 25 days. The last control indicates that the steroids themselves were not used as a carbon source. Interpretation of the growth curves was made by use of the generation time (GT), defined by GT = log 2/k, where k is the growth constant (slope of the growth curve) (4). All values of k were determined by a least squares fit of the data. The effect on growth of different concentrations of the steroids at 25°C. and pH 6.8 are given in Table I. Figure 1 is presented to show examples of representative growth curves. The lowest concentration of each hormone accelerated growth while increasing concentrations generally slowed growth. The highest steroid levels used appear to inhibit growth. The same general effect was noted in experiments at temperatures of 20’ and 30°C. as well as at pH’s of 6.28 and 7.45. The effects noted are reminiscent of a “narcotic effect.” Unfortunately the water solubilities of the steroids prohibit investigations at higher concentrations which could be used to check this point. Cholesterol shows no effect on the generation time. It appears possible that the steroids may be affecting the cell either by “fixing” the cell membrane in a more permeable state as suggested by the mechanism proposed by Eyring and Dougherty (5), or by reaction with specific enzymes in the membrane or in the cytoplasm. Work is continuing to clarify these possibilities. The presence of two different steroids in mammalian experimental systems often results in one steroid modifying the effect of a second (1). Similar effects on this single cell test system are given in Table II. It appears that both testosterone and Compound F modify the growth effect of estrone which is in keeping with observations on mammalian tissues (6). Further it appears that testosterone modifies the growth effect of Compound F, an observation for which there are no data available in mammalian systems for comparison. If the total data presented
LETTERS
TO THE
EDITORS
275
are considered it is seen that no effect and retardation of the generation time are both observed for the same total steroid concentrations. Hence these modifications apparently are not related to the total steroid levels. Neither is the growth effect determined by the presence of the general steroid nucleus as is seen from the fact that cholesterol at those concentrations used, has
0.30 10.20 -
Testosterone Concentration -0.5 mg./liter X 0 mg./liter (control) o 2x) mgJliter
0.001 b-
40
80
120
TIME IN HOURS growth curves of E. gracilis cultured in a testosteronecontaining medium. Each point represents the average of 9 observations (triplicate samples for triplicate runs). The standard deviation for each point is below the plotting precision. Only the log phase of growth was used to calculate generation time. FIG. 1. Representative
TABLE II Combination of Steroids and Mean Generation Time (86’Y?., QH 6.8) Combination of steroids 1. Estrone-Cpd.
2. 3. 4. 5. 6.
F Estrone-Cpd. F Estrone-testosterone Estrone-testosterone Testosterone-Cpd. F Control (no hormone)
Concentrations (rug/liter)
0.1-1.0 1.0-1.0 0.1-2.0 1.0-1.0 1.0-1.0 -
GT (hours) + S.D.
20.84 17.60 21.96 21.14 23.69 18.30
f f f f f -I:
0.59 0.14 1.09 0.87 1.69 0.51
276
LETTERS
TO
THE
EDITORS
no demonstrable effect on the growth of Euglena. Work is at present underway in this laboratory to determine the response of this system to still lower levels of steroids as well as the structural specificity of the steroid required to exert the growth action in this test system. Utilization of these findings with the appropriate modifications may also be of use to those interested in bioassay procedures. REFERENCES
1. ROBERTS, S., AND &EGO, C. M., Physiol. Rev. 33, 593 (1953). 2. CRAMER, M., AND MYERS, J., Arch. Mikrobiol. 17, 384 (1952). 3. EIH-NES, K., SCHELLMAN, J. A., LUMRY, R., AND SAMUELS, L. T., J. Biol. Chem. 206, 411 (1954). 4. HINSHELWOOD, C. N., “The Chemical Kinetics of the Bacterial Cell.” Oxford University Press, London, 1947. 5. EYRING, H., AND DOUGHERTY, T. F., Am. Scientist 43, 457 (1955). 6. ROBSON, J. M., Proc. Sot. Ezptl. Biol. Med. 36, 49 (1936). D. E. BUETOW B. H. LEVEDAHL
Department of Zoology of California, University Los Angeles, California Received October 81, 1967
Transfer of Oxygen-18 during Amino Acid Activation’ Enzymes catalyzing
the activation
of amino acids as measured by the reaction
Amino acid + NHzOH + ATPS -+ Amino acid hydroxamate
+ AMP + pyrophosphate
have been found in animals (1)) plants (2)) and microorganisms (3). Such enzymes also catalyze both an amino acid-dependent exchange of pyrophosphate with ATP, and ATP synthesis from pyrophosphate and synthetic amino acid-AMP compounds (4,5). These findings, plus the lack of occurrence of free intermediates, have been interpreted to mean that the initial step in amino acid activation is the formation of an enzyme-bound amino acid-AMP intermediate (I, 3,4). Evidence for the formation of such an amino acid-AMP compound has now been obtained by determination of the fate of amino acid carboxyl-oxygen during amino acid activation. When tryptophan, labeled in its carboxyl with oxygen-18, is converted to tryptophan hydroxamate by the action of the purified tryptophanactivating enzyme of beef pancreas (6), oxygen-18 appears in the phosphate group of the liberated AMP (Table I). Within experimental error, the amount of oxygen-18 found in the AMP corresponds to that expected for the transfer of one oxygen from the tryptophan carboxyl. In contrast, essentially no oxygen-18 from 1 Supported by grants from the National Science Foundation and the Herman Frasch Foundation. 2 Abbreviations used: ATP, adenosine triphosphate; AMP, adenosine monophosphate.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
73,
%ii-‘i!%
(1957)
LETTERS TO THE EDITORS The Effects of Steroids on the Growth of Euglena
gracitislJ
Steroid effects in mammalian systems have been well investigated, and indeed are sufficiently well understood so that a given program of events can be described in general terms when single steroids or combinations of steroids are introduced into the experimental system. Many steroid hormone effects are reflected in cell proliferation, or “growth,” while combinations of hormones show competition with each other aa reflected in similar criteria (1). The question arises whether it is possible to demonstrate a similar program of events as a result of the introduction of steroids into a suitable single cell system. Such a system must not only show a response to single steroids but should respond to multiple steroids in a pattern that would show some of the interrelationships that have already been demonstrated in fhe mammal. WhiIe admittedly the metazoan system and the single cell system may be very different, such a demonstration could be used to investigate any generalized function of steroids at the cellular level and might even be used to investigate structure requirements of steroids for hormone action. In this communication we wish to report investigations on a single cell system that appears to permit study, by means of growth measurements, of not only a response to characteristic steroid hormones, but also of the interaction or “competition” between combinations of such steroids. The single cell used was a streptomycin-bleached free-living flagellate, Euglena gracilis var. bacillaris. This chlorotic Euglena may be cultivated on a defined medium, does not require sterols or steroids for its growth and avoids the problems encountered with its photosynthetic counterpart. Testosterone, 17-hydroxycorticosterone (Compound F), and estrone were chosen as characteristic steroid hormones while cholesterol was used as a control compound that contains the characteristic steroid nucleus but has no known hormone activity. In these studies growth was measured as optical density at 570 rnp. Cultures were grown in 10 ml. of Cramer-Myers acetate medium (2) contained in 30 ml. screwcap tubes which were shaken in the dark throughout the period of culture growth. All steroid concentrations were within the water solubility range of each steroid (3). The crystalline steroid was added directly to the culture medium during its preparation. Inoculum of 0.1 ml. of organisms from a culture in the log phase of growth was used for each tube. Controls using no hormone, no carbon source and 1 From a dissertation submitted in partial fulfillment of the requirements for the degree of Master of Arts in the University of California, Los Angeles, 1956. * These studies were aided by a contract between the Office of Naval Research, Department of the Navy, and the University of California, Los Angeles, NR 120-336. 273
274
LETTERS
TO
THE
EDITORS
TABLE I Diflerent Concentrations of Steroids and Mean Generation Time- (WC., pH 6.8) GT (hours) C~peAt$$ Ill. I
0 (control) 0.1 0.2 0.5 1.0 1.5 2.0
Estrone
18.80 14.29 16.09 19.02 21.15 -
Testosterone
18.80 15.85 18.72 20.10 21.24
1f -HYdrOWcorticosterone
18.80 17.18 16.38 19.06 21.12
Cholesterol
18.80 18.41 19.03 18.30 -
0 Each dose-response curve was run at least twice and generally three times with triplicate samples each time. Statistical analysis by an analysis of variance shows that the dose-response correlation using the steroid hormones is significant at the 5aj, level in all cases. no hormone, and hormone but no carbon source were run with each experiment. In the latter two controls no growth was observed in 25 days. The last control indicates that the steroids themselves were not used as a carbon source. Interpretation of the growth curves was made by use of the generation time (GT), defined by GT = log 2/k, where k is the growth constant (slope of the growth curve) (4). All values of k were determined by a least squares fit of the data. The effect on growth of different concentrations of the steroids at 25°C. and pH 6.8 are given in Table I. Figure 1 is presented to show examples of representative growth curves. The lowest concentration of each hormone accelerated growth while increasing concentrations generally slowed growth. The highest steroid levels used appear to inhibit growth. The same general effect was noted in experiments at temperatures of 20’ and 30°C. as well as at pH’s of 6.28 and 7.45. The effects noted are reminiscent of a “narcotic effect.” Unfortunately the water solubilities of the steroids prohibit investigations at higher concentrations which could be used to check this point. Cholesterol shows no effect on the generation time. It appears possible that the steroids may be affecting the cell either by “fixing” the cell membrane in a more permeable state as suggested by the mechanism proposed by Eyring and Dougherty (5), or by reaction with specific enzymes in the membrane or in the cytoplasm. Work is continuing to clarify these possibilities. The presence of two different steroids in mammalian experimental systems often results in one steroid modifying the effect of a second (1). Similar effects on this single cell test system are given in Table II. It appears that both testosterone and Compound F modify the growth effect of estrone which is in keeping with observations on mammalian tissues (6). Further it appears that testosterone modifies the growth effect of Compound F, an observation for which there are no data available in mammalian systems for comparison. If the total data presented
LETTERS
TO THE
EDITORS
275
are considered it is seen that no effect and retardation of the generation time are both observed for the same total steroid concentrations. Hence these modifications apparently are not related to the total steroid levels. Neither is the growth effect determined by the presence of the general steroid nucleus as is seen from the fact that cholesterol at those concentrations used, has
0.30 10.20 -
Testosterone Concentration -0.5 mg./liter X 0 mg./liter (control) o 2x) mgJliter
0.001 b-
40
80
120
TIME IN HOURS growth curves of E. gracilis cultured in a testosteronecontaining medium. Each point represents the average of 9 observations (triplicate samples for triplicate runs). The standard deviation for each point is below the plotting precision. Only the log phase of growth was used to calculate generation time. FIG. 1. Representative
TABLE II Combination of Steroids and Mean Generation Time (86’Y?., QH 6.8) Combination of steroids 1. Estrone-Cpd.
2. 3. 4. 5. 6.
F Estrone-Cpd. F Estrone-testosterone Estrone-testosterone Testosterone-Cpd. F Control (no hormone)
Concentrations (rug/liter)
0.1-1.0 1.0-1.0 0.1-2.0 1.0-1.0 1.0-1.0 -
GT (hours) + S.D.
20.84 17.60 21.96 21.14 23.69 18.30
f f f f f -I:
0.59 0.14 1.09 0.87 1.69 0.51
276
LETTERS
TO
THE
EDITORS
no demonstrable effect on the growth of Euglena. Work is at present underway in this laboratory to determine the response of this system to still lower levels of steroids as well as the structural specificity of the steroid required to exert the growth action in this test system. Utilization of these findings with the appropriate modifications may also be of use to those interested in bioassay procedures. REFERENCES
1. ROBERTS, S., AND &EGO, C. M., Physiol. Rev. 33, 593 (1953). 2. CRAMER, M., AND MYERS, J., Arch. Mikrobiol. 17, 384 (1952). 3. EIH-NES, K., SCHELLMAN, J. A., LUMRY, R., AND SAMUELS, L. T., J. Biol. Chem. 206, 411 (1954). 4. HINSHELWOOD, C. N., “The Chemical Kinetics of the Bacterial Cell.” Oxford University Press, London, 1947. 5. EYRING, H., AND DOUGHERTY, T. F., Am. Scientist 43, 457 (1955). 6. ROBSON, J. M., Proc. Sot. Ezptl. Biol. Med. 36, 49 (1936). D. E. BUETOW B. H. LEVEDAHL
Department of Zoology of California, University Los Angeles, California Received October 81, 1967
Transfer of Oxygen-18 during Amino Acid Activation’ Enzymes catalyzing
the activation
of amino acids as measured by the reaction
Amino acid + NHzOH + ATPS -+ Amino acid hydroxamate
+ AMP + pyrophosphate
have been found in animals (1)) plants (2)) and microorganisms (3). Such enzymes also catalyze both an amino acid-dependent exchange of pyrophosphate with ATP, and ATP synthesis from pyrophosphate and synthetic amino acid-AMP compounds (4,5). These findings, plus the lack of occurrence of free intermediates, have been interpreted to mean that the initial step in amino acid activation is the formation of an enzyme-bound amino acid-AMP intermediate (I, 3,4). Evidence for the formation of such an amino acid-AMP compound has now been obtained by determination of the fate of amino acid carboxyl-oxygen during amino acid activation. When tryptophan, labeled in its carboxyl with oxygen-18, is converted to tryptophan hydroxamate by the action of the purified tryptophanactivating enzyme of beef pancreas (6), oxygen-18 appears in the phosphate group of the liberated AMP (Table I). Within experimental error, the amount of oxygen-18 found in the AMP corresponds to that expected for the transfer of one oxygen from the tryptophan carboxyl. In contrast, essentially no oxygen-18 from 1 Supported by grants from the National Science Foundation and the Herman Frasch Foundation. 2 Abbreviations used: ATP, adenosine triphosphate; AMP, adenosine monophosphate.