ChronobiologyInternational Vol. 9, No. 4, pp. 266-268 0 1992 International Society of Chronobiology
A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyeda
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P. Colepicolo, V. C. P. C. Camarero, and *J. Woodland Hastings Instituto de Quimicu, Univrrxidude de Sdo Paulo, Departamento de Bioquimicu, Sdo Puulo. Brazil; and *Department qj"Cellular and Developmental Biology, Hurvurd University, Cambridgt., Mussachuseits, U.S.A.
Summary: The activity of superoxide dismutase in cell-free extracts of Gonyaulux made at different times ofday and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. Key Words: Circadian rhythm-Superoxide dismutase-Gonyuulux polyedra-Unicellular dinoflagellate-Reactive oxygen.
A number of different reactive oxygen species occur as transients in aerobic cells. These active species may include superoxide anion (O,-), hydroxyl radical (OH'), hydrogen peroxide (H,O,), hypochlorite anion (OC1-), and singlet oxygen ('0,)(1,2). The most important metabolites and enzymes responsible for controlling the intracellular levels of such oxygen species are vitamin E, carotenoids, catalase, glutathione peroxidase, and superoxide dismutase. Superoxide dismutases (SOD; EC 1.15.1.1) are metalloenzymes capable of scavenging superoxide anion (3,4). Since some of these species are produced photochemically, they might be more abundant in cells during the daytime. If so, systems for controlling them might also exhibit corresponding variations. In order to investigate this possibility, we measured the SOD activity in extracts of the unicellular marine dinoflagellate Gonyaulaxpolyedra, which is known to display circadian rhythmicity in several properties ( 5 ) , including bioluminescence, swarming motility, photosynthesis, and cell division (Fig. I). SOD activity was found to be highest during the day and to persist under constant conditions, indicating that it is regulated by the circadian clock. Recent results show that circadian changes in bioluminescence are correlated with daily changes in the Received August 17, I99 1 ; accepted with revisions October 10, 199 I , Address correspondence and reprint requests to J. Woodland Hastings, Department of Cellular and Developmental Biology, Harvard University, I6 Divinity Avenue, Cambridge, MA 02 138, U.S.A.
266
CIRCADIAN RHYTHM IN SOD ACTIVITY OF GONYAULAX
FIG. 1. Circadian rhythmicity has been shown in several processes in the unicellular marine dinoflagellate Gonyuulax polyedru. The bioluminescence flashing frequency, glow, luciferase, SOD, nitrate reductase, cell division, photosynthesis, and cell aggregation occur maximally at different phases, as illustrated schematically.
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cellular amounts of luciferase and luciferin-binding protein (LBP) and that a daily pulse in the synthesis is due to translational control (6). However, other than LBP and luciferase, which are synthesized in the early night phase, no other circadian-controlled proteins have been isolated and characterized from such cells. As shown in Fig. 2, the activity of SOD in extracts of G. polyedru exhibits a circadian rhythm, with the peak of activity occurring during the day phase, under conditions of both light-dark cycles and constant dim light. Whether or not this involves de n o w synthesis and destruction during every cycle remains to be determined. However, if confirmed, this regulation constitutes a type previously unknown for SOD; SOD is generally characterized as being inducible by or related to the levels of substrate on which it acts (7,8). If superoxide is generated photochemically, then the cellular level should remain constant in cells kept in constant conditions.
FIG. 2. Circadian oscillation of SOD activity. Gon.vui~luxpolyedru cells were grown in flasks in 1.5-L vol at 19°C under a I 2 h light/l2 h dark (LD) regimen to a cell density of about 1.5 X lo3 cells/ml. Some culture flasks were transferred to constant light (LL). while others were kept on the LD regimen. Starting at time 0, which corresponded to the beginning of the light period on LD, and approximately at the beginning ofthe light phase for the LL cultures, cells were harvested by filtration at the times shown, and extracts were prepared by cell disruption using the French press. SOD activity was determined by a technique involving the inhibition of cvtochrome C reduction. using the hypoxanthine-xanthine oxidase system as the source of superoxide ion, as described by McCord and Fridovich (9). One unit causes 50% inhibition of cytochrome C reduction (I). SOD activity was also measured by an indirect method in which the inhibition of the superoxide-dependent chemiluminescence of luminol was determined (10,l I).The results (not plotted) were closely similar. The protein concentration was measured by the coomassie blue method (Bio-Rad) using the technique of Bradford (12).
7
Chronobiol In(. Vol 9. No. 4, 1992
268
P. COLEPICOLO ET AL.
Acknowledgment: This research was supported by NIH grant GM 19536 NSF grant DMB 8619536 (U.S.A.),and FAPESP, CNPq (Brazil).
Chronobiol Int Downloaded from informahealthcare.com by McMaster University on 02/09/15 For personal use only.
REFERENCES I . Fridovich I. The biology of superoxide and of superoxide dismutases. In: Rogers MAJ, Powers EL, eds. Oxygen and oxyradicals in chemistry and biology. New York: Academic Press, I98 1:197-204. 2. Cadenas E. Biochemistry of oxygen toxicity. Ann Rev Biochem 1989;58:79-110. 3. Lee YM, Friedman DJ, Ayala FJ. Superoxide dismutase: an evolutionary puzzle. Proc Natl Acad Sci USA 1985;82:824-8. 4. Getzoff ED, Tainer JA, Stempien MM, Bell GI, Hallewell RA. Evolution of CuZn superoxide dismutase and the greek key @barrel structure motif. Proteins: .structure,,function and genetics 1989;5:32236. 5. Morse D, Fritz L. Hastings JW. What is the clock? Translational regulation of circadian bioluminescence. Trends Biochem Sci 1990;15:262-5. 6. Morse D, Milos PM, Roux E, Hastings JW. Circadian regulation of the synthesis ofsubstrate binding protein in the Gonyaulax bioluminescent system involves translational control. Proc Null Acad Sci USA 1989;86: 172-6. 7. Goodchild NT, Knock L, Ciborowski L. Effect of superoxide anion (02Jon the survival of Chinese hamster cells. In: Rogers MAJ, Powers EL, eds. Oxygen and oxyradicals in chemistry and biologv. New York: Academic Press, 198 1:649-5 I . 8. Hassan HM, Fridovich I. Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia e l i . J Bucteriol 1977;129: 1574-83. 9. McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969;244:6049-55. 10. Hodgson EK, Fridovich I. Role of superoxide in the chemiluminescence of luminol. Photochem Photobiol 1973;18:45 1-5. 1 1 . Kurfurst M, Ghisla S, Hastings JW. Bioluminescence emission from the reaction of the luciferaseFMN-radical with O,-. Biochemistr.v 1983;22: 152 1-5. 12. Bradford MMA. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochcm 1976;72:248-54.
C'hronobrd hi.Vol 9, No. 4, I992
A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyeda
Chronobiol Int Downloaded from informahealthcare.com by McMaster University on 02/09/15 For personal use only.
P. Colepicolo, V. C. P. C. Camarero, and *J. Woodland Hastings Instituto de Quimicu, Univrrxidude de Sdo Paulo, Departamento de Bioquimicu, Sdo Puulo. Brazil; and *Department qj"Cellular and Developmental Biology, Hurvurd University, Cambridgt., Mussachuseits, U.S.A.
Summary: The activity of superoxide dismutase in cell-free extracts of Gonyaulux made at different times ofday and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. Key Words: Circadian rhythm-Superoxide dismutase-Gonyuulux polyedra-Unicellular dinoflagellate-Reactive oxygen.
A number of different reactive oxygen species occur as transients in aerobic cells. These active species may include superoxide anion (O,-), hydroxyl radical (OH'), hydrogen peroxide (H,O,), hypochlorite anion (OC1-), and singlet oxygen ('0,)(1,2). The most important metabolites and enzymes responsible for controlling the intracellular levels of such oxygen species are vitamin E, carotenoids, catalase, glutathione peroxidase, and superoxide dismutase. Superoxide dismutases (SOD; EC 1.15.1.1) are metalloenzymes capable of scavenging superoxide anion (3,4). Since some of these species are produced photochemically, they might be more abundant in cells during the daytime. If so, systems for controlling them might also exhibit corresponding variations. In order to investigate this possibility, we measured the SOD activity in extracts of the unicellular marine dinoflagellate Gonyaulaxpolyedra, which is known to display circadian rhythmicity in several properties ( 5 ) , including bioluminescence, swarming motility, photosynthesis, and cell division (Fig. I). SOD activity was found to be highest during the day and to persist under constant conditions, indicating that it is regulated by the circadian clock. Recent results show that circadian changes in bioluminescence are correlated with daily changes in the Received August 17, I99 1 ; accepted with revisions October 10, 199 I , Address correspondence and reprint requests to J. Woodland Hastings, Department of Cellular and Developmental Biology, Harvard University, I6 Divinity Avenue, Cambridge, MA 02 138, U.S.A.
266
CIRCADIAN RHYTHM IN SOD ACTIVITY OF GONYAULAX
FIG. 1. Circadian rhythmicity has been shown in several processes in the unicellular marine dinoflagellate Gonyuulax polyedru. The bioluminescence flashing frequency, glow, luciferase, SOD, nitrate reductase, cell division, photosynthesis, and cell aggregation occur maximally at different phases, as illustrated schematically.
T I M E OF D A Y Chronobiol Int Downloaded from informahealthcare.com by McMaster University on 02/09/15 For personal use only.
267
cellular amounts of luciferase and luciferin-binding protein (LBP) and that a daily pulse in the synthesis is due to translational control (6). However, other than LBP and luciferase, which are synthesized in the early night phase, no other circadian-controlled proteins have been isolated and characterized from such cells. As shown in Fig. 2, the activity of SOD in extracts of G. polyedru exhibits a circadian rhythm, with the peak of activity occurring during the day phase, under conditions of both light-dark cycles and constant dim light. Whether or not this involves de n o w synthesis and destruction during every cycle remains to be determined. However, if confirmed, this regulation constitutes a type previously unknown for SOD; SOD is generally characterized as being inducible by or related to the levels of substrate on which it acts (7,8). If superoxide is generated photochemically, then the cellular level should remain constant in cells kept in constant conditions.
FIG. 2. Circadian oscillation of SOD activity. Gon.vui~luxpolyedru cells were grown in flasks in 1.5-L vol at 19°C under a I 2 h light/l2 h dark (LD) regimen to a cell density of about 1.5 X lo3 cells/ml. Some culture flasks were transferred to constant light (LL). while others were kept on the LD regimen. Starting at time 0, which corresponded to the beginning of the light period on LD, and approximately at the beginning ofthe light phase for the LL cultures, cells were harvested by filtration at the times shown, and extracts were prepared by cell disruption using the French press. SOD activity was determined by a technique involving the inhibition of cvtochrome C reduction. using the hypoxanthine-xanthine oxidase system as the source of superoxide ion, as described by McCord and Fridovich (9). One unit causes 50% inhibition of cytochrome C reduction (I). SOD activity was also measured by an indirect method in which the inhibition of the superoxide-dependent chemiluminescence of luminol was determined (10,l I).The results (not plotted) were closely similar. The protein concentration was measured by the coomassie blue method (Bio-Rad) using the technique of Bradford (12).
7
Chronobiol In(. Vol 9. No. 4, 1992
268
P. COLEPICOLO ET AL.
Acknowledgment: This research was supported by NIH grant GM 19536 NSF grant DMB 8619536 (U.S.A.),and FAPESP, CNPq (Brazil).
Chronobiol Int Downloaded from informahealthcare.com by McMaster University on 02/09/15 For personal use only.
REFERENCES I . Fridovich I. The biology of superoxide and of superoxide dismutases. In: Rogers MAJ, Powers EL, eds. Oxygen and oxyradicals in chemistry and biology. New York: Academic Press, I98 1:197-204. 2. Cadenas E. Biochemistry of oxygen toxicity. Ann Rev Biochem 1989;58:79-110. 3. Lee YM, Friedman DJ, Ayala FJ. Superoxide dismutase: an evolutionary puzzle. Proc Natl Acad Sci USA 1985;82:824-8. 4. Getzoff ED, Tainer JA, Stempien MM, Bell GI, Hallewell RA. Evolution of CuZn superoxide dismutase and the greek key @barrel structure motif. Proteins: .structure,,function and genetics 1989;5:32236. 5. Morse D, Fritz L. Hastings JW. What is the clock? Translational regulation of circadian bioluminescence. Trends Biochem Sci 1990;15:262-5. 6. Morse D, Milos PM, Roux E, Hastings JW. Circadian regulation of the synthesis ofsubstrate binding protein in the Gonyaulax bioluminescent system involves translational control. Proc Null Acad Sci USA 1989;86: 172-6. 7. Goodchild NT, Knock L, Ciborowski L. Effect of superoxide anion (02Jon the survival of Chinese hamster cells. In: Rogers MAJ, Powers EL, eds. Oxygen and oxyradicals in chemistry and biologv. New York: Academic Press, 198 1:649-5 I . 8. Hassan HM, Fridovich I. Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia e l i . J Bucteriol 1977;129: 1574-83. 9. McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969;244:6049-55. 10. Hodgson EK, Fridovich I. Role of superoxide in the chemiluminescence of luminol. Photochem Photobiol 1973;18:45 1-5. 1 1 . Kurfurst M, Ghisla S, Hastings JW. Bioluminescence emission from the reaction of the luciferaseFMN-radical with O,-. Biochemistr.v 1983;22: 152 1-5. 12. Bradford MMA. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochcm 1976;72:248-54.
C'hronobrd hi.Vol 9, No. 4, I992
