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Requirement for iron and its effect on ascorbate peroxidase in Euglena gracilis
plan cienc e Plant Science 93 (1993) 25-29
]IN~\IlR ~,( t[ NYIF I~ F'L BI 1~,1[I R'~ IR[ [ \ \ 1 )
Requirement for iron and its effect on ascorbate peroxidase in
Euglena gracilis Takahiro
Ishikawa,
Toru Takeda, Toshio
Shigeru Shigeoka*, Osamu
Hirayama,
Mitsunaga
Department of Food and Nutrition, Kinki University, Nara 631, Japan (Received 10 March 1993; revision received 1 June 1993; accepted 2 June 1993)
Abstract
Euglena gracilis shows an absolute requirement for iron for growth. Iron depletion causes E,glena cells to arrest cell division and the provision of iron allows the iron-deficient cells to return to the normal state. Iron-deficient cells exhaustively take up iron within 1 h of the addition of iron. Incorporated iron exists in a bound form. but not in a free form. The ascorbate peroxidase activity is not found in iron-deficient cells. Lipid peroxides (thiobarbituric acidreactive substances, TBARS) in iron-deficient cells is much higher than those in iron-sufficient cells. The experimental results reported here suggest that iron is involved in the expression of ascorbate peroxidase activity and Euglena ascorbate peroxidase may possess a second important function in the form of a lipid peroxide-scavenging system, in addition to its action of destroying hydrogen peroxide. Key words."Iron requirement; Ascorbate peroxidase; Thiobarbituric acid-reactive substances, TBARS: Euglena gracilis
1. Introduction
Iron (Fe) serves as a prosthetic group for Feproteins, such as hemoglobin, cytochromes and Fe-sulfur proteins [1]. Although Fe is required for the growth of Euglena gracilis [2], the uptake of Fe and the extent and cause of the requirement for Fe have remained unknown [3]. Ascorbate peroxidase is widely distributed in higher plants [4], Euglena [5], green algae [6] and legume root nodules [7] and functions to eliminate * Corresponding author.
hydrogen peroxide generated in vivo. Ascorbate peroxidase is a hemoprotein, like guaiacol peroxidase and cytochrome c, based on absorption spectra of the purified protein [4,5]. Spinach ascorbate peroxidase has been shown to contain a non-heme Fe, which is released from the enzyme in ascorbate-depleted medium under aerobic conditions [8], suggesting that Fe ion may regulate the expression of ascorbate peroxidase. Furthermore, ascorbate peroxidase in Euglena and some cyanobacteria as well as glutathione peroxidase from animal sources can reduce organic hydroperoxides [9,10]. This evidence suggests that ascorbate
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03666-J
26
T. lshikawa et a l . / P l a n t Sci. 93 (1993) 25-29
peroxidase protects the cell membrane and membrane-bound enzymes from damage, loss of integrity and inactivation by the reduction of lipid peroxides generated in vivo. This paper reports the requirement and uptake of Fe by Euglena cells. In order to find a clue to the expression mechanism of ascorbate peroxidase by Fe, we study the effect of Fe on the Euglena ascorbate peroxidase activity. In addition, we determine lipid peroxides in Fe-sufficient and -deficient Euglena cells and discuss the physiological function of ascorbate peroxidase with regard to the lipid peroxide-scavenging system. 2. Materials and methods
2.1. Organism and culture Euglena gracilis Klebs strain z Priingsheim (Fesufficient cells) was grown organotrophically at 26°C for 5 days under illumination (3000 lux) in 150 ml of Koren-Hutner medium containing 50 mg/1 FeSO 4 (NH4)2SO 4. 6H20 which corresponded to 7.1 rag/1 (0.13 mM) of Fe [11]. The cells (14 x 106 cells/ml) grown in this manner were transferred to a basal medium (150 ml) lacking Fe and cultured for 5 days. Subsequently, the cells (1 ml) in stationary phase were again cultured in a Koren-Hutner medium lacking Fe for 5 days to obtain Fe-deficient cells (9 x 106 cells/ml). Fesufficient cells were grown for 5 days in a medium supplemented with 30-, 60- and 100-fold higher concentration of Fe in comparison with that of the original Koren-Hutner medium. These cells, in the stationary phase, represent Fe-excess cells. Cell number was determined with a haemocytometer.
2.2. Determination of iron Euglena cells (wet wt. 0.2 g) were harvested by centrifugation at 3000 x g for 5 min, washed twice with distilled water and resuspended in 5 ml of nitric acid. The concentration of Fe was assayed by measuring absorbance of Fe at 2493 A, using an atomic absorption spectrophotometer (Shimadzu AA-640-12). The relation of peak height to the quantity of Fe was linear up to 100 #mol.
2.3. Assay of iron uptake Fe-deficient cells grown for 5 days, by which
time the stationary phase was reached, were used for assay of iron uptake. 0.13 mM Fe was added to 50 ml of cell culture of Fe-deficient cells. At given intervals, a 5-ml sample was withdrawn and centrifuged at 1000 x g for 5 min to obtain the cell and supernatant fractions. The content of iron in both fractions was determined as described above.
2.4. Subcellular distribution of iron Fe-deficient Euglena cells (wet wt. 0.2 g), which had taken up exogenous Fe for 2 h, were disintegrated by sonication (10 kHz, 2 min) in 3 ml of 50 mM Tris-HC1 buffer (pH 6.9) and centrifuged at 500 x g for 3 min to remove the cell debris. The cell homogenate was centrifuged at 10 000 x g for 10 min to obtain the 10 000 × g-precipitate and then the supernatant fraction was ultracentrifuged at 100 000 x g for 30 min. Subsequently, the 100 000 x g-supernatant fluid was chromatographed on a Sephadex G-25 column (1.8 x 45 cm) equilibrated with 50 mM Tris-HC1 buffer (pH 6.9) at a flow rate of 24 ml h -l and the eluant was collected (1.2-ml fraction).
2.5. Assays of ascorbate peroxidase and lipid peroxides Fe-sufficient, -deficient and -excess Euglena cells grown to the stationary phase were used for assays of ascorbate peroxidase activity and lipid peroxides. Crude extracts of Euglena cells were prepared and assayed for ascorbate peroxidase as described previously [9]. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS) [12]. Five ml of 1% (w/v) trichloroacetic acid was added to Euglena cells (wet wt. 1 g). The mixture was sonicated (10 kHz) for a total of 1 min with two intervals of 30-s each and centrifuged at 10 000 x g for 10 min. The supernatant fraction was used to determine TBARS. Each value represents the mean of four assays ± S.D. 3. Results and discussion
Fig. 1 shows growth curves of Fe-sufficient, -deficient and -excess (30-fold) Euglena cells. Each culture reached stationary phase in 5 days. The cell
27
T. Ishikawa et al. / Plant Sci. 93 (1993) 25-29
~
8
~
4 2 0
i
i
i
0
i
i
2
i
4 Time
L
i
6
8
(day)
Fig. 1. Growth curves of Fe-suficient,-deficient and -excess Euglena cells. I , Fe-sufficient: O, Fe-deficient; I , Fe-excess (30-fold). The dashed line (D) shows the growth curve of Fedeficient cells after the addition of 7.1 mg/l Fe. Each experimental point represents the mean of four assays (coefficient of variation < 5%)
growth of Fe-deficient cells decreased to 66% in comparison with that of Fe-sufficient cells. When 0.13 mM Fe, which corresponds to that of Fe-sufficient cells, was added to Fe-deficient cultures, cell division commenced and the cell number reached the same level as that of Fesufficient cells within 2 days. The content of Fe in E. gracilis in stationary phase under each growth condition was determined. Fe-sufficient cells contained 0.18 ± 0.02 mg of Fe per 109 cells, indicating that 35.5% of the amount of Fe present
©
0.5
© ~
0.4 s v
4
0.3
3
0.2
2
o
~ 0.1
O
1
2
3
4
Time (hr)
Fig. 2. Changes in Fe levels in Fe-supplemented cells and medium. O, Fe in cells; 0, Fe in medium. Fe (0.13 mM) was added to the Fe-deficient cells.
in the original medium was taken up in the Fesufficient cells. In contrast, Fe was not detected in Fe-deficient cells grown for 4 - 6 days, showing that Fe-deficient cells contain little iron or extremely low amounts beyond the limit of the Fe measurement. These results demonstrate that Fe is absolutely required for the growth of E. gracilis. When cells were grown in a medium supplemented with a 30-fold higher Fe concentration (213 mg/l) than that of Fe-sufficient cells, there was no change of the cell growth up to, and including stationary phase. The same result was obtained from cells grown in a medium containing 60- or 100-fold higher Fe concentration (data not shown). The cellular content of Fe in 30-, 60- and 100-fold Feexcess cells was 0.38 ± 0.02, 0.78 -4- 0.03 and 1.13 ± 0.03 rag, respectively, per 10 9 cells, indicating that 2.7%, 2.7% and 2.4"/0 of the original amount of Fe supplied was accumulated in the cells. These results suggest that Euglena possesses a regulatory system that prevents the incorporation of a large amount of external Fe so that the cellular concentration of Fe is maintained at relatively low levels. When 0.13 mM Fe was added to Fe-deficient cultures, Fe was incorporated linearly and peaked at 1 h (Fig. 2). The rate of Fe uptake in Fedeficient cells was calculated as 2.5/~mol/h per 109 cells. The subcellular distribution of Fe taken up for 2 h was examined by the differential centrifugation method (Table 1). Of Fe, 69% was located in the 10 000 × g-precipitated fraction, 4% in the 100 000 × g-precipitated fraction and 18% in the 100 000 × g-supernatant fraction. Subsequently, the 100 000 × g supernatant was chromatographed on a Sephadex G-25 column. As shown in Fig. 3, Fe was predominantly eluted in the high molecular weight fractions. These data demonstrate that Fe incorporated into the Euglena cells exists mostly in a bound form, not in a free-form. It has been reported that free Fe becomes toxic to many cellular components since it is involved in the reaction of superoxide anion and hydrogen peroxide, that is, in the Fenton reaction, to produce hydroxyl radicals which are the most reactive species of active oxygen [13]. Accordingly, the fact that Euglena cells limit the cellular level of Fe and avoid accumulation of free cellular Fe seems to be
28
T. lshikawa et al. / Plant Sci. 93 (1993) 25-29
12
I
"E
8
,,=
0
10
20
Fraction number
10 8
4
30
40
(1.4 ml/tube)
Fig. 3. Elution pattern of Fe in the 100 000 x g-supernatant fraction by column chromatography on Sephadex G-25. The 100 000 x g-supernatant fraction obtained by differential centrifugation was applied to a Sephadex G-25 column and eluted with a 50-mM Tris-HC1 buffer (pH 6.9). See Materials and methods for details. O, Fe: O, protein.
a general strategy for the suppression of the oxidative stress produced by free Fe. It is well known that Fe plays a critical role in the expression of heme and non-heme proteins of living organisms [1]. Ascorbate peroxidase is a hemoprotein li'ke guaiacol peroxidase and cytochrome c. By dialysis of the purified spinach ascorbate peroxidase against 50 mM phosphate buffer (pH 7.6), the Fe content of the enzyme decreased to about half and the activity was lost, because ascorbate peroxidase also contains one atom of non-heme Fe, which is involved in the stability of the enzyme [8,14]. In this context, it is
Table 1 Distribution of Fe content and protein in subcellular fractions of Fe-deficient cells with added 0.13 mM Fe for 2 h Fraction
Protein (t~g)
Crude homogenate 10 000 x g Ppt 100 000 x g Ppt 100 000 x g Sup
190.0 + 6.8 (100%) a 65.0 + 5.1 (100%) 92.0 + 5.6 (46) 24.8 4- 2.1 (13) 51.5 ~ 4.5 (27)
Fe content (,g)
44.7 ± 3.5 (69) 2.9 + 0.2 (4) 11.5 + 1.8 (18)
Preparation of crude homogenate and differential centrifugation was carried out as described in Materials and methods. Each value represents the mean of three assays + S.D. aPercent distribution in parentheses.
interesting to investigate how Fe affects the activity of ascorbate peroxidase in Fe-sufficient and -deficient Euglena cells. Fe-sufficient cells in the stationary phase contain 75.3 4- 2.3 ~mol/min per 10 9 cells of ascorbate peroxidase activity, which corresponds to the value reported previously [151 (Table 2). In contrast, no enzyme activity was found in Fe-deficient cells grown for 4-6 days. We have previously reported that 50 ~M ferrous sulfate is necessary to prevent extractable ascorbate peroxidase from inactivation [9]. However, the enzyme activity was not recovered by incubation of the crude extract prepared from Fe-deficient cells with 50 I~M ferrous sulfate (data not shown). These results suggest that Fe fed exogenously is incorporated into Euglena cells and then is involved in the expression of ascorbate peroxidase activity. Detailed study of the mechanism of appearance of ascorbate peroxidase after the addition of iron to Fe-deficient cells is under study. Ascorbate peroxidase in Euglena [91 and some cyanobacteria [10] reduces various organic hydroperoxides as well as hydrogen peroxide, suggesting that this enzyme, like glutathione peroxidase, protects the cell membrane and prevents inactivation of membrane-bound enzymes from damage by lipid peroxides generated endogenously from unsaturated fatty acids. Euglena contains a large quantity of polyunsaturated fatty acids [16]. As mentioned above, the ascorbate peroxidase activity was not observed in Fe-deficient cells. These results indicate that Fe-deficient Euglena cells may be useful organisms for elucidating in detail the physiological role of ascorbate peroxidase in the lipid peroxide-scavenging system. Thus TBARS was determined in Fe-sufficient, -deficient and excess Euglena cells grown to stationary phase (Table 2). TBARS in Fe-deficient cells was about 2.5-fold greater than that in Fe-sufficient cells. The increase in TBARS in Fe-deficient cells seems to stem from the depletion of ascorbate peroxidase activity. The TBARS in Fe-excess cells was 2.3fold higher than that in Fe-sufficient cells. In this case, ascorbate peroxidase may be unable to remove large amounts of lipid peroxides from lipid peroxidation, caused by hydroxyl radicals produced by Fe accumulated in Fe-excess cells. The findings that the activity of ascorbate peroxidase in
T. lshikawa et aL /Plant Sci. 93 (1993) 25-29 Table 2 Effect of iron on AsAP activity and lipid peroxides in Euglena. Fe-suficient,-deficient and -excess Euglena cells grown in the stationary phase were used for assays for ascorbate peroxidase activity (AsAP) and lipid peroxides (TBARS). Cells
Fe-sufficient Fe-deficient Fe-excess
29 5
6
AsAP activity (#mol/109cells/min)
TBARS (#mol/109cells)
7
75.3 + 2.3 n.d. 77.5 + 3.1
6.2 + 0.6 15.8 + 2.1 14.3 4- 1.9
8
Each value represents the mean of four assays + S.D.n.d., not detected. 9
Fe-excess cells did not increase with the cellular concentration of Fe (Table 2) and that 73% of Fe incorporated into the cells was distributed in the membranes (Table 1) support this view. Thus we suggest that Euglena ascorbate peroxidase possesses a lipid peroxide scavenging function as a second function, in addition to its action of destroying hydrogen peroxide in vivo. 4. References 1 2
3
4
P. Aisen and I. Listowsky, Iron transport and storage proteins. Ann. Rev. Biochem., 49 (1980) 357-393. B.D. Knezek and R.H. Maier, Influence of level and source of Fe on growth and nutrient content of Euglena gracilis. Soil Sci. Plant Anal., 2 (1971) 37-44. E.S. Kempner, Stimulation and inhibition of the metabolism and growth of Euglena gracilis, in: D.E. Buetow (Ed.), The Biology of Euglena, Vol. 3, Academic Press, New York, 1982, pp. 197-252. K. Asada, Ascorbate peroxidase--a hydrogen peroxidescavenging enzyme in plants. Physiol. Plant., 85 (1992) 235-241.
10
11
12 13 14
15
16
S. Shigeoka, Y. Nakano, and S. Kitaoka, Metabolism of hydrogen peroxide in Euglena gracilis z by L-ascorbic acid peroxidase. Biochem. J., 186 (1980) 377-380. Y.W. Kow, D.A. Smyth and M. Gibbs, Oxidation of reduced pyridine nucleotide by a system using ascorbate and hydrogen peroxide from plants and algae. Plant Physiol., 69 (1982) 72-76. D.A. Dalton, F.J. Hanus, S.A. Russell and H.J. Evans, Purification, properties and distribution of ascorbate peroxidase in legume root nodules. Plant Physiol., 83 (1987) 789-794. Y. Nakano and K. Asada, Purification of ascorbate peroxidase in spinach chloroplsts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol., 28 (1987) 131-140. S. Shigeoka, Y. Nakano and S. Kitaoka, Purification and some properties of L-ascorbic acid-specific peroxidase in Euglena gracilis Z. Arch. Biochem. Biophys., 201 (1980) 121-127. E. Tel-Or, M.E. Huflejt and L. Packer, Hydroperoxide metabolism in cyanobacteria. Arch. Biochem Biophys., 246 (1986) 396-402. L.E. Koren and S.H. Hutner, High-yield media for photosynthesizing Euglena gracilis Z. J. Protozool., 14 (1967) suppl. 17. J.A. Buege and S.D. Aust, Microsomal lipid peroxidation. Methods Enzymol., 52 (1978) 302-310. B. Halliwell and J.M.C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University Press, 1985. G-X. Chen and K. Asada, Ascorbate peroxidase in tea leaves: occurrence of two isozymes and the differences in their enzymatic and molecular properties. Plant Cell Physiol., 30 (1989) 987-998. S. Shigeoka, R. Yasumoto, T. Onishi, Y. Nakano and S. Kitaoka, Properties of monodehydroascorbate reductase and dehydroascorbate reductase and their participation in the regeneration of ascorbate in Euglena gracilis. J. Gen. Microbiol., 133 (1987) 227-232. D. Hulanicka, J. Erwin and K. Bloch, Lipid metabolism of Euglena gracilis. J. Biol. Chem., 239 (1964) 2778-2787.
]IN~\IlR ~,( t[ NYIF I~ F'L BI 1~,1[I R'~ IR[ [ \ \ 1 )
Requirement for iron and its effect on ascorbate peroxidase in
Euglena gracilis Takahiro
Ishikawa,
Toru Takeda, Toshio
Shigeru Shigeoka*, Osamu
Hirayama,
Mitsunaga
Department of Food and Nutrition, Kinki University, Nara 631, Japan (Received 10 March 1993; revision received 1 June 1993; accepted 2 June 1993)
Abstract
Euglena gracilis shows an absolute requirement for iron for growth. Iron depletion causes E,glena cells to arrest cell division and the provision of iron allows the iron-deficient cells to return to the normal state. Iron-deficient cells exhaustively take up iron within 1 h of the addition of iron. Incorporated iron exists in a bound form. but not in a free form. The ascorbate peroxidase activity is not found in iron-deficient cells. Lipid peroxides (thiobarbituric acidreactive substances, TBARS) in iron-deficient cells is much higher than those in iron-sufficient cells. The experimental results reported here suggest that iron is involved in the expression of ascorbate peroxidase activity and Euglena ascorbate peroxidase may possess a second important function in the form of a lipid peroxide-scavenging system, in addition to its action of destroying hydrogen peroxide. Key words."Iron requirement; Ascorbate peroxidase; Thiobarbituric acid-reactive substances, TBARS: Euglena gracilis
1. Introduction
Iron (Fe) serves as a prosthetic group for Feproteins, such as hemoglobin, cytochromes and Fe-sulfur proteins [1]. Although Fe is required for the growth of Euglena gracilis [2], the uptake of Fe and the extent and cause of the requirement for Fe have remained unknown [3]. Ascorbate peroxidase is widely distributed in higher plants [4], Euglena [5], green algae [6] and legume root nodules [7] and functions to eliminate * Corresponding author.
hydrogen peroxide generated in vivo. Ascorbate peroxidase is a hemoprotein, like guaiacol peroxidase and cytochrome c, based on absorption spectra of the purified protein [4,5]. Spinach ascorbate peroxidase has been shown to contain a non-heme Fe, which is released from the enzyme in ascorbate-depleted medium under aerobic conditions [8], suggesting that Fe ion may regulate the expression of ascorbate peroxidase. Furthermore, ascorbate peroxidase in Euglena and some cyanobacteria as well as glutathione peroxidase from animal sources can reduce organic hydroperoxides [9,10]. This evidence suggests that ascorbate
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03666-J
26
T. lshikawa et a l . / P l a n t Sci. 93 (1993) 25-29
peroxidase protects the cell membrane and membrane-bound enzymes from damage, loss of integrity and inactivation by the reduction of lipid peroxides generated in vivo. This paper reports the requirement and uptake of Fe by Euglena cells. In order to find a clue to the expression mechanism of ascorbate peroxidase by Fe, we study the effect of Fe on the Euglena ascorbate peroxidase activity. In addition, we determine lipid peroxides in Fe-sufficient and -deficient Euglena cells and discuss the physiological function of ascorbate peroxidase with regard to the lipid peroxide-scavenging system. 2. Materials and methods
2.1. Organism and culture Euglena gracilis Klebs strain z Priingsheim (Fesufficient cells) was grown organotrophically at 26°C for 5 days under illumination (3000 lux) in 150 ml of Koren-Hutner medium containing 50 mg/1 FeSO 4 (NH4)2SO 4. 6H20 which corresponded to 7.1 rag/1 (0.13 mM) of Fe [11]. The cells (14 x 106 cells/ml) grown in this manner were transferred to a basal medium (150 ml) lacking Fe and cultured for 5 days. Subsequently, the cells (1 ml) in stationary phase were again cultured in a Koren-Hutner medium lacking Fe for 5 days to obtain Fe-deficient cells (9 x 106 cells/ml). Fesufficient cells were grown for 5 days in a medium supplemented with 30-, 60- and 100-fold higher concentration of Fe in comparison with that of the original Koren-Hutner medium. These cells, in the stationary phase, represent Fe-excess cells. Cell number was determined with a haemocytometer.
2.2. Determination of iron Euglena cells (wet wt. 0.2 g) were harvested by centrifugation at 3000 x g for 5 min, washed twice with distilled water and resuspended in 5 ml of nitric acid. The concentration of Fe was assayed by measuring absorbance of Fe at 2493 A, using an atomic absorption spectrophotometer (Shimadzu AA-640-12). The relation of peak height to the quantity of Fe was linear up to 100 #mol.
2.3. Assay of iron uptake Fe-deficient cells grown for 5 days, by which
time the stationary phase was reached, were used for assay of iron uptake. 0.13 mM Fe was added to 50 ml of cell culture of Fe-deficient cells. At given intervals, a 5-ml sample was withdrawn and centrifuged at 1000 x g for 5 min to obtain the cell and supernatant fractions. The content of iron in both fractions was determined as described above.
2.4. Subcellular distribution of iron Fe-deficient Euglena cells (wet wt. 0.2 g), which had taken up exogenous Fe for 2 h, were disintegrated by sonication (10 kHz, 2 min) in 3 ml of 50 mM Tris-HC1 buffer (pH 6.9) and centrifuged at 500 x g for 3 min to remove the cell debris. The cell homogenate was centrifuged at 10 000 x g for 10 min to obtain the 10 000 × g-precipitate and then the supernatant fraction was ultracentrifuged at 100 000 x g for 30 min. Subsequently, the 100 000 x g-supernatant fluid was chromatographed on a Sephadex G-25 column (1.8 x 45 cm) equilibrated with 50 mM Tris-HC1 buffer (pH 6.9) at a flow rate of 24 ml h -l and the eluant was collected (1.2-ml fraction).
2.5. Assays of ascorbate peroxidase and lipid peroxides Fe-sufficient, -deficient and -excess Euglena cells grown to the stationary phase were used for assays of ascorbate peroxidase activity and lipid peroxides. Crude extracts of Euglena cells were prepared and assayed for ascorbate peroxidase as described previously [9]. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS) [12]. Five ml of 1% (w/v) trichloroacetic acid was added to Euglena cells (wet wt. 1 g). The mixture was sonicated (10 kHz) for a total of 1 min with two intervals of 30-s each and centrifuged at 10 000 x g for 10 min. The supernatant fraction was used to determine TBARS. Each value represents the mean of four assays ± S.D. 3. Results and discussion
Fig. 1 shows growth curves of Fe-sufficient, -deficient and -excess (30-fold) Euglena cells. Each culture reached stationary phase in 5 days. The cell
27
T. Ishikawa et al. / Plant Sci. 93 (1993) 25-29
~
8
~
4 2 0
i
i
i
0
i
i
2
i
4 Time
L
i
6
8
(day)
Fig. 1. Growth curves of Fe-suficient,-deficient and -excess Euglena cells. I , Fe-sufficient: O, Fe-deficient; I , Fe-excess (30-fold). The dashed line (D) shows the growth curve of Fedeficient cells after the addition of 7.1 mg/l Fe. Each experimental point represents the mean of four assays (coefficient of variation < 5%)
growth of Fe-deficient cells decreased to 66% in comparison with that of Fe-sufficient cells. When 0.13 mM Fe, which corresponds to that of Fe-sufficient cells, was added to Fe-deficient cultures, cell division commenced and the cell number reached the same level as that of Fesufficient cells within 2 days. The content of Fe in E. gracilis in stationary phase under each growth condition was determined. Fe-sufficient cells contained 0.18 ± 0.02 mg of Fe per 109 cells, indicating that 35.5% of the amount of Fe present
©
0.5
© ~
0.4 s v
4
0.3
3
0.2
2
o
~ 0.1
O
1
2
3
4
Time (hr)
Fig. 2. Changes in Fe levels in Fe-supplemented cells and medium. O, Fe in cells; 0, Fe in medium. Fe (0.13 mM) was added to the Fe-deficient cells.
in the original medium was taken up in the Fesufficient cells. In contrast, Fe was not detected in Fe-deficient cells grown for 4 - 6 days, showing that Fe-deficient cells contain little iron or extremely low amounts beyond the limit of the Fe measurement. These results demonstrate that Fe is absolutely required for the growth of E. gracilis. When cells were grown in a medium supplemented with a 30-fold higher Fe concentration (213 mg/l) than that of Fe-sufficient cells, there was no change of the cell growth up to, and including stationary phase. The same result was obtained from cells grown in a medium containing 60- or 100-fold higher Fe concentration (data not shown). The cellular content of Fe in 30-, 60- and 100-fold Feexcess cells was 0.38 ± 0.02, 0.78 -4- 0.03 and 1.13 ± 0.03 rag, respectively, per 10 9 cells, indicating that 2.7%, 2.7% and 2.4"/0 of the original amount of Fe supplied was accumulated in the cells. These results suggest that Euglena possesses a regulatory system that prevents the incorporation of a large amount of external Fe so that the cellular concentration of Fe is maintained at relatively low levels. When 0.13 mM Fe was added to Fe-deficient cultures, Fe was incorporated linearly and peaked at 1 h (Fig. 2). The rate of Fe uptake in Fedeficient cells was calculated as 2.5/~mol/h per 109 cells. The subcellular distribution of Fe taken up for 2 h was examined by the differential centrifugation method (Table 1). Of Fe, 69% was located in the 10 000 × g-precipitated fraction, 4% in the 100 000 × g-precipitated fraction and 18% in the 100 000 × g-supernatant fraction. Subsequently, the 100 000 × g supernatant was chromatographed on a Sephadex G-25 column. As shown in Fig. 3, Fe was predominantly eluted in the high molecular weight fractions. These data demonstrate that Fe incorporated into the Euglena cells exists mostly in a bound form, not in a free-form. It has been reported that free Fe becomes toxic to many cellular components since it is involved in the reaction of superoxide anion and hydrogen peroxide, that is, in the Fenton reaction, to produce hydroxyl radicals which are the most reactive species of active oxygen [13]. Accordingly, the fact that Euglena cells limit the cellular level of Fe and avoid accumulation of free cellular Fe seems to be
28
T. lshikawa et al. / Plant Sci. 93 (1993) 25-29
12
I
"E
8
,,=
0
10
20
Fraction number
10 8
4
30
40
(1.4 ml/tube)
Fig. 3. Elution pattern of Fe in the 100 000 x g-supernatant fraction by column chromatography on Sephadex G-25. The 100 000 x g-supernatant fraction obtained by differential centrifugation was applied to a Sephadex G-25 column and eluted with a 50-mM Tris-HC1 buffer (pH 6.9). See Materials and methods for details. O, Fe: O, protein.
a general strategy for the suppression of the oxidative stress produced by free Fe. It is well known that Fe plays a critical role in the expression of heme and non-heme proteins of living organisms [1]. Ascorbate peroxidase is a hemoprotein li'ke guaiacol peroxidase and cytochrome c. By dialysis of the purified spinach ascorbate peroxidase against 50 mM phosphate buffer (pH 7.6), the Fe content of the enzyme decreased to about half and the activity was lost, because ascorbate peroxidase also contains one atom of non-heme Fe, which is involved in the stability of the enzyme [8,14]. In this context, it is
Table 1 Distribution of Fe content and protein in subcellular fractions of Fe-deficient cells with added 0.13 mM Fe for 2 h Fraction
Protein (t~g)
Crude homogenate 10 000 x g Ppt 100 000 x g Ppt 100 000 x g Sup
190.0 + 6.8 (100%) a 65.0 + 5.1 (100%) 92.0 + 5.6 (46) 24.8 4- 2.1 (13) 51.5 ~ 4.5 (27)
Fe content (,g)
44.7 ± 3.5 (69) 2.9 + 0.2 (4) 11.5 + 1.8 (18)
Preparation of crude homogenate and differential centrifugation was carried out as described in Materials and methods. Each value represents the mean of three assays + S.D. aPercent distribution in parentheses.
interesting to investigate how Fe affects the activity of ascorbate peroxidase in Fe-sufficient and -deficient Euglena cells. Fe-sufficient cells in the stationary phase contain 75.3 4- 2.3 ~mol/min per 10 9 cells of ascorbate peroxidase activity, which corresponds to the value reported previously [151 (Table 2). In contrast, no enzyme activity was found in Fe-deficient cells grown for 4-6 days. We have previously reported that 50 ~M ferrous sulfate is necessary to prevent extractable ascorbate peroxidase from inactivation [9]. However, the enzyme activity was not recovered by incubation of the crude extract prepared from Fe-deficient cells with 50 I~M ferrous sulfate (data not shown). These results suggest that Fe fed exogenously is incorporated into Euglena cells and then is involved in the expression of ascorbate peroxidase activity. Detailed study of the mechanism of appearance of ascorbate peroxidase after the addition of iron to Fe-deficient cells is under study. Ascorbate peroxidase in Euglena [91 and some cyanobacteria [10] reduces various organic hydroperoxides as well as hydrogen peroxide, suggesting that this enzyme, like glutathione peroxidase, protects the cell membrane and prevents inactivation of membrane-bound enzymes from damage by lipid peroxides generated endogenously from unsaturated fatty acids. Euglena contains a large quantity of polyunsaturated fatty acids [16]. As mentioned above, the ascorbate peroxidase activity was not observed in Fe-deficient cells. These results indicate that Fe-deficient Euglena cells may be useful organisms for elucidating in detail the physiological role of ascorbate peroxidase in the lipid peroxide-scavenging system. Thus TBARS was determined in Fe-sufficient, -deficient and excess Euglena cells grown to stationary phase (Table 2). TBARS in Fe-deficient cells was about 2.5-fold greater than that in Fe-sufficient cells. The increase in TBARS in Fe-deficient cells seems to stem from the depletion of ascorbate peroxidase activity. The TBARS in Fe-excess cells was 2.3fold higher than that in Fe-sufficient cells. In this case, ascorbate peroxidase may be unable to remove large amounts of lipid peroxides from lipid peroxidation, caused by hydroxyl radicals produced by Fe accumulated in Fe-excess cells. The findings that the activity of ascorbate peroxidase in
T. lshikawa et aL /Plant Sci. 93 (1993) 25-29 Table 2 Effect of iron on AsAP activity and lipid peroxides in Euglena. Fe-suficient,-deficient and -excess Euglena cells grown in the stationary phase were used for assays for ascorbate peroxidase activity (AsAP) and lipid peroxides (TBARS). Cells
Fe-sufficient Fe-deficient Fe-excess
29 5
6
AsAP activity (#mol/109cells/min)
TBARS (#mol/109cells)
7
75.3 + 2.3 n.d. 77.5 + 3.1
6.2 + 0.6 15.8 + 2.1 14.3 4- 1.9
8
Each value represents the mean of four assays + S.D.n.d., not detected. 9
Fe-excess cells did not increase with the cellular concentration of Fe (Table 2) and that 73% of Fe incorporated into the cells was distributed in the membranes (Table 1) support this view. Thus we suggest that Euglena ascorbate peroxidase possesses a lipid peroxide scavenging function as a second function, in addition to its action of destroying hydrogen peroxide in vivo. 4. References 1 2
3
4
P. Aisen and I. Listowsky, Iron transport and storage proteins. Ann. Rev. Biochem., 49 (1980) 357-393. B.D. Knezek and R.H. Maier, Influence of level and source of Fe on growth and nutrient content of Euglena gracilis. Soil Sci. Plant Anal., 2 (1971) 37-44. E.S. Kempner, Stimulation and inhibition of the metabolism and growth of Euglena gracilis, in: D.E. Buetow (Ed.), The Biology of Euglena, Vol. 3, Academic Press, New York, 1982, pp. 197-252. K. Asada, Ascorbate peroxidase--a hydrogen peroxidescavenging enzyme in plants. Physiol. Plant., 85 (1992) 235-241.
10
11
12 13 14
15
16
S. Shigeoka, Y. Nakano, and S. Kitaoka, Metabolism of hydrogen peroxide in Euglena gracilis z by L-ascorbic acid peroxidase. Biochem. J., 186 (1980) 377-380. Y.W. Kow, D.A. Smyth and M. Gibbs, Oxidation of reduced pyridine nucleotide by a system using ascorbate and hydrogen peroxide from plants and algae. Plant Physiol., 69 (1982) 72-76. D.A. Dalton, F.J. Hanus, S.A. Russell and H.J. Evans, Purification, properties and distribution of ascorbate peroxidase in legume root nodules. Plant Physiol., 83 (1987) 789-794. Y. Nakano and K. Asada, Purification of ascorbate peroxidase in spinach chloroplsts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol., 28 (1987) 131-140. S. Shigeoka, Y. Nakano and S. Kitaoka, Purification and some properties of L-ascorbic acid-specific peroxidase in Euglena gracilis Z. Arch. Biochem. Biophys., 201 (1980) 121-127. E. Tel-Or, M.E. Huflejt and L. Packer, Hydroperoxide metabolism in cyanobacteria. Arch. Biochem Biophys., 246 (1986) 396-402. L.E. Koren and S.H. Hutner, High-yield media for photosynthesizing Euglena gracilis Z. J. Protozool., 14 (1967) suppl. 17. J.A. Buege and S.D. Aust, Microsomal lipid peroxidation. Methods Enzymol., 52 (1978) 302-310. B. Halliwell and J.M.C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University Press, 1985. G-X. Chen and K. Asada, Ascorbate peroxidase in tea leaves: occurrence of two isozymes and the differences in their enzymatic and molecular properties. Plant Cell Physiol., 30 (1989) 987-998. S. Shigeoka, R. Yasumoto, T. Onishi, Y. Nakano and S. Kitaoka, Properties of monodehydroascorbate reductase and dehydroascorbate reductase and their participation in the regeneration of ascorbate in Euglena gracilis. J. Gen. Microbiol., 133 (1987) 227-232. D. Hulanicka, J. Erwin and K. Bloch, Lipid metabolism of Euglena gracilis. J. Biol. Chem., 239 (1964) 2778-2787.