superoxide dismutase in scenedesmus obliquus

Planta (1985)~63:405 410 Planta �9 Springer-Verlag 1985

Superoxide dismutase in Scenedesmus obliquus Effect of growth conditions and initial characterization

Mark J. Redmond*, Alan R. McEuen** and Roy Powls Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK

Abstract. In heterotrophically grown Scenedesmus obliquus, the specific activity of superoxide dismutase (SOD; EC 1.15.1.1) declined when glucose was abundant, increased as it was depleated, and remained steady at a high level when it was absent. Transition to autotrophic growth produced only a small (20% over 5 d) increase in specific activity above the values obtained in dark-grown cells after glucose and starch-reserve depletion. This small, but consistent, increase did, however, parallel a similar increase in photosynthetic capacity. Poly- acrylamide-gel electrophoresis showed the ex- istence of nine isoenzymes of SOD. The three major and one of the minor isoenzymes were present in all extracts while three minor isoenzymes were found only in autotrophically grown cells and two only in heterotrophically grown cells. Character- ization studies indicated that two of the major isoenzymes are dimeric FeSODs the other is a tetrameric MnSOD, and of the minor isoenzymes, two are dimeric FeSODs and four are dimeric MnSODs.

Key words: Glucose effect - Mehler reaction - Su- peroxide dismutase isoenzymes - Scenedesmus.

Introduction

Superoxide, the one-electron reduction product of molecular oxygen, is produced in many biological systems, and it, or products derived from it, can

Present addresses: * Department of Biochemistry, Medical Sciences Building,

University of Alberta, Edmonton, Alberta, T6G 2H7, Can- ada

** Department of Biology, Rivers State College of Education, PMB 5047, Port Harcourt, Nigeria

Abbreviation : SOD = superoxide dismutase

be highly toxic (Halliwell 1977; Fridovich 1979). Accordingly, virtually all aerobic cells possess superoxide dismutase (SOD), which catalyzes the dis- proportionation of superoxide to hydrogen peroxide and oxygen

2 0 ~ . + H + -+ 0 2 q - H 2 0 2

The amount of SOD is not fixed, but varies in response to environmental and metabolic conditions (Hassan and Fridovich 1977a, b; Friedberg et al. 1979; Tanaka and Sugahara 1980). During photosynthesis, superoxide is produced by the reaction of oxygen with the reduced products ofpho- tosystem I (the Mehler reaction) (Harbour and Bolton 1975; Asada and Nakano 1978). In Scene- desmus obliquus, the Mehler reaction is greatest upon initial illumination, but then declines to a steady rate, which carries on indefinitely (Radmer and Kok 1976). As S. obliquus can be grown either heterotrophically or autotrophically, we decided to investigate the effects of the different growth conditions on SOD levels and isoenzyme composition, and in particular to monitor changes following the transfer from growth in the dark to growth in the light.

There are three types of SOD, distinguished by their prosthetic metals: the cuprozinc enzyme, which is inhibited by cyanide, the manganese and iron enzymes, which are not. The cuprozinc enzyme, although widespread in higher plants and animals is generally absent in algae (Lumsden and Hall 1975; Asada etal. 1977; Henry and Hall 1977), the only reported exceptions being Spiro- gyra and Chara (Henry and Hall 1977). Although MnSOD has been purified from a red alga (Misra and Fridovich 1977) and FeSOD from Euglena (Kanematsu and Asada 1979), there has been no characterization of the prosthetic metals of any cyanide-resistant SOD from the division Chloro-

406 M.J. Redmond et al. : Superoxide dismutase in Scenedesmus

phyta. We therefore include in this report the iden- tification of the prosthetic metal of the SOD's of Scenedesmus obliquus.

Material and methods

Chemicals. Nitro blue tetrazolium (NBT) was obtained from Calbiochem C.P. Laboratories, Bishops Stortford, U K ; horse heart ferricytochrome c (type III), xanthine oxidase (grade III), and diethylenetriaminepentaacetic acid (DTPA) from Sigma Chemical Co., Poole, Dorset, U K ; and glucose oxidase- ABTS reagent (GOD-Perid) from Boehringer, Mannheim, FRG.

Plant materials. Cultures of Scenedesmus obliquus Gaffron D3 (strain 276/6a; Culture Collection of Algae and Protozoa, Cam- bridge, UK) were grown in an orbital incubator at 25~ in media prepared according to Kessler et al. (1957). The complete medium contained 0.5% glucose, 0.2% yeast extract, and inorganic salts. In some instances, either the yeast extract was omitted (glucose minimal medium) or the glucose was omitted (yeast minimal medium). The medium used for autotrophic growth contained only the inorganic salts.

Cells were harvested by centrifugation at 1600 g for 10 rain, washed twice with extraction buffer (50 mM potassium phosphate, 0.1 mM DTPA pH 7.8) and suspended to a final volume of 10 ml. Cells were disrupted in a Braun (Melsungen, FRG) homogeniser (10 ml beads, 0.25 mm diameter) for 3 min using a cycle of 25 s CO 2 cooling/5s no cooling. After removal of the glass beads by filtration, the cell-free extract was centrifuged for 10 min at 48000g. The supernatant fraction was stored frozed until use.

Enzyme assays. Superoxide dismutase was assayed by the cyto- chrome c - xanthine oxidase method of McCord and Fridovich (1969) as modified by Keele et al. (1971). Protein was determined by the tannin micromethod of Mejbaum-Katzenellenbo- gen and Dobryszycka (1959). Glucose was assayed by glucose oxidase and ABTS according to the manufacturer's instruc- tions. Photosynthetic capacity was determined using an oxygen electrode to measure the rate of oxygen evolution for an illuminated cell suspension, which had been adjusted to give an ab- sorbance of 0.5 at 550 nm. All measurements being made at 30 ~ C under saturating light conditions.

Electrophoresis. Polyacrylamide-gel electrophoresis was per- formed according to Maurer (1971). Superoxide dismutase was visualized by NBT (Beauchamp and Fridovich 1971) and protein by Coomassie Blue R (Weber and Osborn 1969). Molecular weights were determined by the method of Hendrick and Smith (1968) with transferrin, bovine serum albumin, MnSOD from Bacillus stearothermophilus, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and cr as standards.

Partial purification of SOD. Cells (110 g wet weight) were suspended in 0.3 M 2-amino-2-(hydroxmethyl)-l,3-propanediol (Tris)-HC1, pH 7.5, containing 0.05% 2-mercaptoethanol to a final volume of 200 ml, and disrupted by grinding with glass beads (150 ml beads, 0.25 mm diameter) for 10 rain in a Dyno- mill homogeniser (W.A. Bachofen, Basle, Switzerland). Effi- cient cooling was achieved by an ice-salt mixture continually circulating the breaking chamber. After filtration and centrifugation, ammonium sulphate was added to 40% saturation. The precipitated proteins were removed by centrifugation and the ammonium-sulphate concentration of the supernatant fraction was increased to 90% saturation. After centrifugation the pellet was resuspended in 0.04 M Tris-HC1, pH 7.5, containing 0.05%

2-mercaptoethanol and dialysed against the same buffer over- night at 4 ~ C. The sample was then applied to a (20 cm long, 2.5 cm diameter) column of DE 52 ion-exchange cellulose (Whatman, Springfield Mdl, Kent, UK) equilibrated with 0.05 M Tris-HC1, pH 7.5, and eluted with a linear salt gradient made by mixing 220 ml of 0.05 M Tris-HC1, pH 7.5, with 220 ml of 1.0 M Tris-HC1, pH 7.5. Fractions (6.4 ml) were col- lected using a flow rate of 30 ml h - 1.

Results

The effect of growth conditions on SOD content. Cultures were grown heterotrophically in the dark in complete medium at 25 ~ C. The specific activity of SOD varied markedly during the growth of the seed culture (Fig. 1 a). When glucose was omitted from the growth medium, SOD levels remained high (Fig. 1 b). However, when glucose was present, regardless of whether or not yeast extract was present as well, SOD specific activity decreased while glucose was abundant, and then increased when glucose levels fell (Fig. 1 a, c). Thus, S. obliquus appears to exhibit a glucose effect similar to that reported for Escherichia coli (Hassan and Fri- dovich 1977 b).

Heterotrophically grown S. obliquus was harvested, the cells washed and resuspended in 1-1 batches of autotrophic medium to an optical den- sity of 0.67 at 550 nm. This algal suspension was starved of its starch reserves by incubating aerobi- cally in the dark for 4 d (Fig. 2). During this dark incubation period, the specific activity of the SOD consistently attained a value between 50 and 60 McCord-Fridovich units per mg protein regardless

o f the initial specific activity in the inoculum. Upon illumination of the starved cells, the specific activity of SOD increased gradually over a period of 5 d, reaching levels 20-30% higher than those at the beginning of illumination (Fig. 2). This increase in the SOD specific activity consistently par- alleled an increase in the photosynthetic capacity of the intact cells, as defined by gmol 0 2 evolved min -1 for a cell suspension of fixed Ass o under defined conditions.

Isoenzymes of SOD. Examination of the cell-free extract by polyacrylamide-gel electrophoresis showed the presence of nine isoenzymes: six in he- terotropically grown cells and seven in autotrophically grown cells (Fig. 3). (The isoenzymes have been numbered by the International Union of Bio- chemistry convention that the most rapidly migrat- ing isoenzyme is No. 1). The three major isoenzymes (Nos. 5, 6, and 7) were present in all extracts and in approximately the same relative propor- tions. The minor isoenzyme No. 4 was also usually

M.J. Redmond et a l . Superoxide dismutase in Scenedesmus 407

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present, but as its intensity increased during stor- age, and was often absent in fresh preparations, it is most likely derived from isoenzyme No. 5. The minor isoenzymes Nos. 1, 2, and 3 were found only in autotrophically grown cells, and isoenzymes Nos. 8 and 9 (also minor) were found only in heterotrophically grown cells. Changes in isoenzyme composition could not be monitored during the transition from heterotrophic to autotrophic growth because, in the relatively dilute extracts of the early stages of autotrophic growth, only the major isoenzymes were detectable.

408

Table 1. Summary of characterisation studies of SOD isoenzymes from S. obliquus

Iso- Inhibition Inacti- Metal Pros- Molec- en- by vation restoring thetic ular zyme KCN" by activity (con- weight d

H202 b to apo- clusion) enzyme c

1 No effect No effect Mn 50000 2 No effect No effect - Mn 41000 3 No effect Inactivated Fe 45 000 4 No effect Inactivated Fe Fe 45 000 5 No effect Inactivated Fe Fe 44000 6 No effect Inactivated Fe Fe 45000 7 No effect No effect Mn Mn 83000 8 No effect No effect - Mn 42000 9 No effect No effect - Mn 36000

By method of Beauchamp and Fridovich (1971) b By method of Britton et al. (1978)

By method of Kirby et al. (1980) a By method of Hendrick and Smith (1968)

M.J. Redmond et al. : Superoxide dismutase in Scenedesmus

Table 2. Determination of prosthetic metals of SOD isoenzymes of S. obliquus by method of Kirby et al. (1980). Samples were initially dialysed against 2.5 M guanidinium chloride, 20 mM 8-hydroxyquinoline, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 5mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1, pH 3.8, and then split into three portions: (a) dialysis against 5 mM Tris-HC1, 0.1 mM EDTA, pH 7.8 ('denatured'), (b) dialysis against 0.1 mM MnC12, 5 mM Tris-HC1, pH 7.8 and (c) dialysis against 1.0 mM Fe(NH4)2(SO4) a

Sample Superoxide dismutase (U/mg protein)

Un- "Dena- Dialysed Dialysed treated tured" against against

Mn2+ Fe 2+

Fraction A 421 562 1253 560 (isoenzymes 8, 9)

Fraction B 471 0 51 526 (isoenzymes 4,5,6,7)

The results o f charac te r iza t ion studies are sum- mar ized in Table 1. Cyanide did not inhibit any i soenzyme on the gel, nor did it have any effect in the solut ion assay for SOD, in ag reement with Asada et al. (1977). H y d r o g e n peroxide inact ivates F e S O D but not M n S O D , so the results o f soaking gels in 5 m M H 2 0 2 pr ior to staining for SOD activity (Brit ten et al. 1978) indicate tha t i soenzymes Nos . 1, 2, 7, 8, and 9 conta in m a n g a n e s e and tha t i soenzymes Nos . 3, 4, 5 and 6 conta in iron. The molecular weights de te rmined by the m e t h o d o f Hendr i ck and Smith (1968) are in good agreement with previous ly publ ished values for M n S O D and F e S O D f r o m other sources, and indicate tha t i soenzyme 7 is te t ramer ic whereas all the others are dimeric.

Fu r the r charac te r iza t ion was achieved by the dena tu ra t ion- recons t i tu t ion p rocedure of K i r b y et al. (1980), using samples o f enzyme which had been par t ia l ly purif ied by d ie thy laminoethyl cellulose c h r o m a t o g r a p h y . The S O D eluted f r o m the co lumn in two peaks : f rac t ion A did not b ind to the co lumn and conta ined isoenzymes 8 and 9, while f rac t ion B was eluted by the salt gradient and con ta ined i soenzymes 4, 5, 6 and 7. Dialysis o f f rac t ion A against 2.5 M guan id in ium chloride, 20 m M 8-hydroxyquinol ine , and 0.1 m M ethylene- d iaminete t raace t ic acid, p H 3.8, did not result in any loss o f activity (Table 2). A l though subsequent dialysis against M n 2 § doub led activity, Fe 2 § had no effect. F rac t ion B lost all activity af ter the first dialysis, bu t M n 2§ res tored isoenzyme 7, while Fe 2§ res tored isoenzymes 4, 5, and 6 (Fig. 4). A new band also appea red in the Fe- t rea ted samples

Fig. 4. Disc etectrophoresis of fraction B stained for SOD activity. a Untreated sample; b, denatured sample; c, sample for dialysis against Mn 2 § ; d, sample after dialysis against Fe 2 +

M.J. Redmond et al. : Superoxide dismutase in Scenedesmus 409

with a mobility intermediate to isoenzymes 5 and 6. One possible explanation is that it is a hybrid composed of one subunit of isoenzyme 5 and one subunit of isoenzyme 6 (compare Dougherty et al. 1978).

Discussion

The effect of glucose as a carbon source for heterotrophically grown S. obliquus is very similar to the situation with E. coli observed by Hassan and Fri- dovich (1977 b). In trypticase soy-yeast extract sup- plemented with glucose, SOD specific activity declined when glucose was abundant and increase sharply as it was depleted. As in S. obliquus, this increase began before all the glucose in the medium was depleted. Hassan and Fridovich (1977b) con- cluded that SOD levels dropped in response to the lower level of O2. produced during fermentative catabolism of glucose and that SOD levels rose as the cells switched to the oxidative catabolism of organic acids. However, Bdellovibrio stolpii also shows a similar fall, then rise, in SOD specific activity when grown in batch culture (Von Stein et al. 1982), even though B. stolpii is an obligate aerobe and apparently unable to utilize glucose, Von Stein et al. (1982) postulated that during rapid growth, it is more efficient for the organism to repair or replace damaged cellular components, than to ex- pend effort synthesizing SOD.

The situation in S. obliquus is more akin to the situation in E. coli because, in the absence of glucose, there was no drop in SOD specific activity, even though the growth rates on yeast minimal medium and glucose minimal medium were com- parable.

The most notable aspect of the transition from the stationary phase of heterotrophic growth to autotrophic growth is that there is only a small change in SOD specific activity. Indeed, the level of SOD in stationary-phase, heterotrophically grown cells is very high in comparison with other organisms (Asada et al. 1979; McCord et al. 1971). One possible explanation is that since the Mehler reaction (and hence, O~-. production) is at its maximum in the first few minutes of illumination (Radmer and Kok 1976), the SOD activity would also have to be at its maximum during this period. Rather than wait for a burst of 0 2 to trigger SOD biosynthesis, the cell synthesizes the SOD before- hand to minimize the toxic effects of the Mehler reaction. Furthermore, the 20-30% increase in SOD specific activity in the light was always paral- leled by a similar increase in photosynthetic capacity, prompting one to speculate that the biosynthe-

sis of chloroplast SOD and photosynthetic reaction centres might be coordinated. However, the presence of glucose effect complicates the picture, and more research is clearly needed.

In contrast to the results reported here, Euglena was found to undergo a threefold increase in SOD specific activity upon transfer from heterotropic to autotrophic growth (Asada et al. (1977)). How- ever, Euglena is colourless when grown heterotrophically in the dark and takes several hours to 'green up ' and achieve maximum photosynthetic capacity after transfer to the light, whilst S. obliquus D3 is green and develops a fully competent photosynthetic apparatus even when grown in darkness.

Of the three major SOD isoenzymes in S. obliquus, two are dimeric FeSODs and the other is a tetrameric MnSOD. Until its discovery in Eug- lena gracilis (Kanematsu and Asada 1979), FeSOD was thought to be restricted to prokaryotes. Its presence in a typical representative of the Chloro- phyta indicates that its presence in lower eukaryotes may be quite widespread. It is generally absent in higher eukaryotes, although Salin and Bridges (1980) have found FeSOD as a minor isoenzyme in Brassica campestris. Tetrameric MnSOD is commonly found in eukaryotes, especially in mitochondria. Whether or not isoenzyme No. 7 is so localised in S. obliquus could not be determined because the use of the Braun or Dynomill cell homogeniser, which is necessit- ated by the tough cell wall, is too severe a means of cell disruption to permit isolation of intact or- ganelles.

Of the six minor SOD isoenzymes, four contain manganese and two contain iron, and all are prob- ably dimeric. The multiplicity of isoenzymes re- mains a puzzle, as does the appearance of isoenzymes Nos. 1, 2, and 3 and the disappearance of isoenzymes Nos. 8 and 9 upon transition to autotrophic growth. Quantitatively, they appear to be too minor to account for the 20-30% increase in specific activity during prolonged autotrophic growth.

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410 M.J. Redmond et al. : Superoxide dismutase in Scenedesmus

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Received 12 June; accepted 14 August 1984

superoxide dismutase in scenedesmus obliquus

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