Manganese Superoxide Dismutase and Higher Plant Chloroplasts: A Reappraisal of a Controverted Cellular Localization

Manganese Superoxide Dismutase and Higher Plant Chloroplasts: A Reappraisal of a Controverted Cellular Localization

JOSE M. PALMA, LUISA M. SANDAUO and LUIS A. DEL RIo

Unidad de Bioqufmica Vegetal, Estaci6n Experimental del Zaidfn, C.S.I.c., Prof. Albareda 1, 18008 Granada, Spain

Received March 19, 1986 . Accepted April 17, 1986

Summary

In this work, the disputed localization of manganese-containing superoxide dismutase (EC 1.15.1.1) in higher plant chloroplasts was reexamined. Chloroplasts were isolated from Pisum sativum L. leaves by three different procedures, they were further purified by centrifugation in Percoll density-gradients (21- 60 %) and percentages of chloroplast intactness of 78 - 85 % were obtained. When different batches of intact and hypotonically disrupted chloroplasts, obtained by the three isolation methods, were preincubated with Triton X-I00 and subjected to polyacrylamide gel electrophoresis, only a copperzinc-containing SOD isozyme was found which appeared to be partially bound to thylakoid membranes, but no Mn-containing SOD activity was detected. A band of Mn-SOD was only visible when isolated chloroplasts were not resuspended and washed with the isolation medium, but this band was always removed after washing the chloroplasts, suggesting that the Mn-SOD was due to a contamination by other cellular fractions. Likewise, highly purified intact chloroplasts exclusively contained Cu,Zn-SOD activity, and Mn-SOD activity was clearly absent. The possible reasons for the present controversy on the localization of Mn-SOD in chloroplasts are analyzed with the conclusion that the Mn-SOD activity previously reported by some authors is probably due to different compounds able to react with O2- - radicals occurring within chloroplasts, and thus showing nonspecific or mimic Mn-SOD activity.

Key words: Pisum sativum L.; chloroplasts; cuprozinc superoxide dismutase; manganese superox· ide dismutase; superoxide dismutase.

Introduction

Superoxide dismutases (EC 1.15.1.1) are a group of metalloenzymes that catalyze the disproportionation of superoxide free radicals (02--), generated in different cellular loci by univalent reduction of molecular oxygen, to H 20 2 and O2 (Fridovich, 1983). These enzymes generally occur in three different molecular forms containing either Fe, Mn, or Cu plus Zn as prosthetic metals, and appear to play an important role in protecting cells against the indirect deleterious effects of superoxide free radicals (Rabinowitch and Fridovich, 1983). In higher plants, cuprozinc- and manganesecontaining SODs are widely destributed (Asada et aI., 1980; Rabinowitch and Frido-

Abbreviation list: SOD, superoxide dismutase; Cu,Zn-SOD, cupro-zinc-containing superoxide dismutase; Mn-SOD, manganese-containing superoxide dismutase; Fe-SOD, iron-containing superoxide dismutase; NBT, nitro blue tetrazolium.

J Plant Physiol. Vol. 125. pp_ 427-439 (1986)

428 JosE M. PALMA, LUISA M. SANDALIO and LUIS A. DEL Rio

vich, 1983). Fe-SODs were thought to be present only in certain plant families (Gingkoaceae, Nymphaceae, and Cruciferae) (Bridges and Salin, 1981) but more recently they have also been found in leaves of lemon (Sevilla et al., 1984), beans and tomatoes (Kwiatowski and Kaniuga, 1984).

Copperzinc-containing superoxide dismutases from several plants have been purified and characterized, including those from pea seeds (Sawada et al., 1972), pea leaves (Duke and Salin, 1983), wheat germ (Beauchamp and Fridovich, 1973), spinach leaves (Asada et aI., 1973), kidney bean leaves (Kono et al., 1979), mung bean seedlings (Reddy and Venkaiah, 1984), and cotyledons and shoots of lentils (Federico et al., 1985). This type of superoxide dismutase has been found located mainly within chloroplasts (Asada et al., 1973; Foster and Edwards, 1980; Jackson et al., 1978; Salin and Bridges, 1980 b) but they also have been reported present in the cytosol (Baum and Scandalios, 1979; Foster and Edwards, 1980; Sandalio and Del RIo, 1986), in mitochondria (Arron et al., 1976; Jackson et aI., 1978; Salin and Bridges, 1981; Sandalio and Del Rio, 1986), and in glyoxysomes, a type of specialized plant peroxisome (Sandalio and Del Rio, 1986).

Iron-containing plant superoxide dismutases have been purified and characterized from several procaryotic organisms (Steinman, 1982) and from the eucaryotic green alga Euglena gracilis (Kanematsu and Asada, 1979; Lengfelder and Elstner, 1979), as well as from leaves of mustard (Salin and Bridges, 1980 a), waterlily (Salin and Bridges, 1982), tomato (Kwiatowski et al., 1985) and Gingko biloba (Duke and Salin, 1985), and Fe-SODs appear to be located only in chloroplasts (Duke and Salin, 1985; K wiatowski et al., 1985; Salin and Bridges, 1980 b).

As for manganese-containing SODs, these enzymes have been characterized from a photosynthetic bacterium and a red alga (Steinman, 1982), and in higher plants the enzyme from pea leaves has been fully characterized (Sevilla et aI., 1980; Fernandez et aI., 1982; Sevilla et al., 1982), and very recently the characterization of a Mn-SOD from spinach leaves has been reported (Hayakawa et al., 1985). Mn-SOD has been found present in mitochondria from different plant species including spinach Oackson et al., 1978), maize (Baum and Scandalios, 1979), Jerusalem artichoke (Arron et al., 1976), mustard (Salin and Bridges, 1981), several C3 and C4 plants (Foster and Edwards, 1980) and watermelon (Sandalio and Del RIO, 1986), but the presence of MnSOD in pea peroxisomes (Del Rio et al., 1983) and in watermelon glyoxysomes has also been reported (Sandalio and Del RIO, 1986).

However, the question of the intracellular location of Mn-SOD in chloroplasts is a disputed subject. In contrast to blue-green and green alga where Mn-SOD has been described in chloroplast thylakoids only (Kanematsu and Asada, 1979; Okada et aI., 1979; Lengfelder and Elstner, 1979), higher plants represent a more conflicting picture. The presence of Mn-SOD in chloroplast lamellar membranes from spinach leaves was first claimed in the last decade (Lumsden and Hall, 1974) though this evidence was soon refuted by other workers (Elstner and Heupel, 1975). The absence of Mn-SOD was reported in chloroplasts isolated from maize and several C3 and C4 plants (Baum and Scandalios, 1979; Foster and Edwards, 1980), and also in chloroplasts from pea leaves by applying immunocytochemical techniques to intact protoplasts (Del Rio et al., 1983).

j. Plant Physiol. Vol. 125. pp. 427-439 (1986)

Mn superoxide dismutase and chloroplasts 429

In spite of all these negative experimental evidences! new claims were made on the presence of a Mn-SOD in chloroplast thylakoids (Asada et aI., 1980; Hayakawa et aI., 1984), and very recently the hypothesis of a chloroplast location for Mn-SOD has been supported by the report of the characterization of a thylakoid-bound Mn-superoxide dismutase in chloroplasts (Hayakawa et al., 1985).

In order to try to clarify this conflicting picture, we decided to reexamine this question and study the superoxide dismutase(s) present in chloroplasts which were isolated by three different procedures and were further purified by centrifugation in Percoll density-gradients. This paper shows the unequivocal absence of Mn-SOD activity in all chloroplast samples studied and the single presence of a copperzinccontaining superoxide dismutase in these organelles.

Materials and Methods

Plant material

Pea seeds (Pisum sativum L., cv. Lincoln) were grown either in perlite or in aerated full-nutrient solutions, in a growth chamber, Conviron PGW-36, under optimum conditions as described elsewhere (Del Rio et al., 1985 a). Leaves were harvested 15 to 20 days after planting.

Isolation 0/ chloroplasts

All operations were performed at 0 - 4 °C and homogenization was carried out in a Sorvall Omnimixer by blending at high speed for 2 - 3 seconds. Three different isolation methods were used:

1) A modification of the method of Cockburn et al. (1968). Leaves were homogenized in a medium containing OAM sorbitol, 10mM N04P207, 5mM MgCh, and 2mM Na-ascorbate at pH 6.5 (leaf to medium ratio 1: 4; w/v). The homogenate was filtered through eight layers of nylon cloth and centrifuged at 2200g for 60 seconds (whole time). The pellet was gently suspended in OAM sorbitol, ImM MgCh, 2mM EDTA, 50mM Hepes buffer (PH 6.7), ImM MnCh, and was again centrifuged under the same conditions. The washed chloroplasts were carefully resuspended in a small volume of the latter medium.

2) The method of Nakatani and Barber (1977). Leaves were homogenized in a medium containing 0.33 M sorbitol, 0.2 mM MgCh, in 20 mM Mes buffer, pH 6.5 (1: 4; w/v). The homogenate was filtered through eight layers of nylon cloth and was centrifuged at 2200 g for 90 seconds (whole time), and the pellet was gently suspended in 0.33 M sorbitol adjusted to pH 7.5 with Tris (cation free medium). The suspension was centrifuged at 2200 g for 60 seconds (whole time) and the washed chloroplasts were carefully suspended in a small volume of cation free medium.

3) A modification of the method described by Schwitzguebel and Siegenthaler (1984) for the isolation of peroxisomes and mitochondria from spinach leaves. The homogenization medium contained 0.35M mannitol, 30mM Mops buffer (PH 7.5), 4mM cysteine, ImM EDTA, and 0.2 % BSA (leaf to medium ratio 1: 4; w/v). Homogenates were filtered through four layers of nylon cloth and were centrifuged at 2200g for 30 seconds (whole time), and the pellet was suspended in 0.3 M mannitol, 20 mM Mops buffer (PH 7.2), 1 mM EDT A, 0.2 % BSA. The suspension was centrifuged at 2200g for 30 seconds (whole time) and the washed chloroplasts were carefully resuspended in a small volume of the same medium.

Purification 0/ chloroplasts

Chloroplasts from pea leaves were purified by density-gradient centrifugation in discontinuous gradients of Percoll (Sigma Chemical Co.) by the method described by Schwitzguebel

J. Plant Physiol. Vol. 125. pp. 427-439 (1986)

430 JosE M. PALMA, LUISA M. SANDALIO and LUIS A. DEL Rio

and Siegenthaler (1984) for the purification of peroxisomes and mitochondria from spinach leaves. All operations were performed at 0-4 0c.

Chloroplasts were first isolated by method no. 3 described in the previous section. Volumes of washed chloroplast suspensions of 2.5 ml, containing about 6 - 12 mg chlorophyll, were layered on top of discontinuous density-gradients of Percoll (21- 60 %; v/v) with the following composition: 6ml of 60% Percoll; 14ml of 45%; 8ml of 27% and 8ml of 21 %. All Percoll solutions contained 0.25M sucrose, 10mM Mops buffer (PH 7.2), 0.2% BSA, and to the 21 % and 27 % Percoll layers propane-l,2-diol was added to a 0.1 M final concentration. The gradients with the samples were centrifuged at 13,000 g for 30 min either in a Sorvall centrifuge, using a 55-34 rotor, or in a Beckman ultracentrifuge using a 60 Ti rotor. The gradients were eluted by carefully pumping from the bottom of the tubes. The transmittance of eluted samples was monitored at 280 nm, and they were finally collected in volumes of about 2 mI.

Assays

Total superoxide dismutase activity was determined by the ability of SOD to inhibit nitrite formation from hydroxylamine by the O 2. - -generating system, xanthine oxidase plus xanthine (Elstner and Heupel, 1976), with the following modifications: sulphanilic acid and cx-naphthylamine were replaced by identical concentrations of sulphanilamide and naphthylethylene diamine diHCI, respectively. Total SOD activity in gradient fractions was also assayed by the ferricytochrome c method using xanthine/xanthine oxidase as the source of superoxide radicals (McCord and Fridovich, 1969). SOD isozymes were individualized by polyacrylamide disc gel electrophoresis on 10% acrylamide gels at pH 8.9 according to Davis (1964). Prior to electrophoresis, organelle samples were diluted 2-5-fold with 50mM phosphate buffer, pH 7.8, containing Triton X-I00 (0.2 % final concentration) and stirred to solubilize bound superoxide dismutase(s}. SODs were localized on the gels by the method of the NBT reduction by O 2.

radicals generated photochemically (Weisiger and Fridovich, 1973) and the activity bands were recorded using a Vernon PHI-6 densitometer.

In fractions from Percoll density-gradient centrifugation, the SOD activity determinations were carried out quickly and samples were appropriately diluted in order to reverse the Percoll inhibitory action on SODs (Del RIo et al., 1985 b). Catalase, cytochrome c oxidase, and chlorophyll were used as markers for peroxisomes, mitochondria, and chloroplasts, respectively. Catalase activity was determined according to Aebi (1984), and cytochrome c oxidase was assayed as described by Schnarrenberger et al. (1971). Chlorophyll and proteins were determined according to Arnon (1949) and Bradford (1976), respectively. Measurements of the density of Percoll solutions were carried out using an Atago refractometer.

The intactness of isolated and purified chloroplasts was determined by the method of the ferricyanide reduction, either measuring the ferricyanide-dependent O 2 evolution with a Clark oxygen electrode or by following spectrophotometrically the change in optical density at 410 nm (Lilleyet al., 1975). In chloroplasts purified by Percoll density-gradient centrifugation, the organelle intactness was also estimated by determining the activity of photosynthetic fructose-1,6-bisphosphatase in the isolated fractions (Uzaro et aI., 1974).

Results

Pea leaves (Pisum sativum L.) contain three electrophoretically distinct superoxide dismutases, a slower-moving Mn-containing SOD and two Cu,Zn-containing SODs, named I and II, in order of increasing mobility (Del Rio et aI., 1978). The isozyme Mn-SOD has been fully characterized (Sevilla et aI., 1980; Sevilla et aI., 1982; Fernandez et aI., 1982), and the two pea leaf Cu,Zn-SODs have been purified and characterized recently (Duke and Salin, 1983). The different batches of chloroplasts isolated from pea leaves by the three distinct procedures used had intactness percentages in

J. Plant Pbysiol. Vol. 125. pp. 427-439 (1986)


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Fig. 1: Superoxide dismutase activity of chloroplasts isolated from Pisum sativum L.leaves. Intact and broken chloroplasts were subjected to polyacrylamide gel electrophoresis on 10 % gels and stained for SOD activity by a photochemical method. Broken chloroplasts were prepared from intact ones by dilution with hypotonic medium, and after 30 min at 4 °C, the chlorophyll-containing membranes were recovered by centrifugation at 5,000 g for 10 min and suspended in a small volume of hypotonic medium. Intact and disrupted chloroplasts were incubated, prior to electrophoresis, with Triton X-100 at a 0.2 % final concentration. Samples applied to the gels contained 130-180 JLg chlorophyll. 1, intact chloroplasts; 2, broken chloroplasts; 3, intact chloroplasts stained in the presence of 2 mM NaCN; 4, broken chloroplasts stained in the presence of 2 mM NaCN.

the range 22 - 57 %. When intact and hypotonically disrupted chloroplasts, obtained using the three isolation methods, were preincubated with Triton X-I00 and subjected to polyacrylamide gel electrophoresis, only a CN-sensitive band of activity was detected (Fig. 1) which corresponded by its electrophoretic mobility to isozyme Cu,Zn-SOD II, and no CN-resistant Mn-containing SOD activity was detected. These results were repeatedly obtained with different batches of chloroplasts isolated by the three methods used. A band of Mn-SOD activity was only visible when isolated chloroplasts were not resuspended and washed with the isolation medium, but this band was always removed after washing the chloroplasts. This indicates that the Mn-SOD band was a contamination probably due to mitochondria and/or peroxisomes where this type of SOD has been demonstrated to be present in different plant species (Arron et al., 1976; Baum and Scandalios, 1979; Del Rio et al., 1983; Foster and Edwards, 1980; Jackson et al., 1978; Salin and Bridges, 1981; Sandalio and Del Rio, 1986) and this assumption was corroborated by the activities of cytochrome c oxidase and catalase determined in the crude chloroplasts which were considerably reduced after washing these organelles with isolation medium.

The presence of superoxide dismutase activity was also studied in chloroplasts purified by centrifugation in Percoll density-gradients as shown in Fig. 2. Two highdensity bands containing intact chloroplasts (fr. 3 -6 and 7 -14) were detected in addition to a lower-density band containing broken chloroplasts (fr. 15-20). The two higher-density chloroplast bands (1.094g x ml- I and 1.075 g x ml- I

, respectively)


432 JosE M. PALMA, LUISA M. SANDAUO and LUIS A. DEL Rio

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Washed chloroplasts from pea leaves isolated by the method of Schwitzguebel and Siegenthaler (1984) were applied to a discontinuous density-gradient of Percoll (21- 60 %) and centrifuged in an angle rotor at 13,000 g for 30 min. The chloroplast suspension applied to the gradient (2.5 ml) contained about 12 mg chlorophyll and 130 mg protein. Fractions of 2 ml were collected by pumping from the bottom of the tube. 0, superoxide dismutase; f:::., chlorophyll; ., cytochrome c oxidase; 0, catalase; ., refractive index.

showed percentages of organelle intactness in the range 78 - 85 % and also coincided with two peaks of the chloroplast enzyme fructose-l,6-bisphosphatase (data not shown). The splitting of the intact chloroplasts into two bands after centrifugation in discontinuous Percoll density-gradients has also been reported recently by Mousdale and Coggins (1985) for the same plant species in the course of a work on the shikimate-pathway enzymes.

Two peaks of superoxide dismutase activity were associated with the two bands of intact chloroplasts, and the level of enzyme activity was in approximately the ratio of the chlorophyll content. The higher-density chloroplast band (1.094 g x ml- I ) was found to be rather-pure whereas the other band was contaminated with peroxisomes



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8 Fig.3: Distribution of superoxide dismutase isozymes in highly purified intact chloroplasts from pea leaves. Washed chloroplasts, and highly intact chloroplasts isolated and purified as described in Fig. 2, were diluted with 50 mM phosphate buffer, pH 7.8, containing Triton X-lOO and stirred to solubilize bound superoxide dismutase. Sample volumes of 100 JLl were subjected to polyacrylamide gel electrophoresis, and the gels were stained for SOD activity and scanned with a densitometer. A, crude leaf homogenate (15JLg chlorophyll); B, washed chloroplasts (120JLg chlorophyll); C, highly purified intact chloroplasts (30 JLg chlorophyll).

and mitochondria, as evidenced by the co-eluting peaks of the corresponding organelle markers. Thus, the higher-density intact chloroplast peak was not cross-contaminated and, accordingly, was a very appropriate material for studying the presence of superoxide dismutase(s) by polyacrylamide gel electrophoresis (Fig. 3). All of the gradient fractions defining each chloroplast peak were analyzed and it was found that the band of pure and highly intact chloroplasts (fractions 3 -6) exclusively contained isozyme CU,Zn-SOD II and the levels of isozyme activity detected in acrylamide gels followed the ratio of the chlorophyll peak. No Mn-containing superoxide dismutase activity was detected in pure and intact chloroplasts and this isozyme only appeared



from fraction 12 onwards, coinciding with the increase in cytochrome c oxidase activity, the mitochondrial marker used.

Discussion

The results reported here clearly show the absence of Mn-containing superoxide dismutase activity in chloroplasts isolated from Pisum sativum L. leaves by three different procedures, and also in highly intact chloroplasts purified by Percoll densitygradient centrifugation. In all of the different batches of chloroplasts assayed only a copperzinc-containing SOD isozyme was found which appears to be partially bound to thylakoid membranes as shown by the results from hypotonic shock experiments. Similar results have been reported for chloroplasts isolated from spinach leaves (Asada et al., 1973; Elstner and Heupel, 1975), several C3 and C4 plants (Foster and Edwards, 1980), and maize (Baum and Scandalios, 1979), where the only SOD found was the isozyme Cu,Zn-SOD.

In recent experiments carried out in our laboratory, peroxisomes and mitochondria were purified from pea leaves by density-gradient centrifugation and manganese-containing superoxide dis mutase activity was found to be present both in mitochondrial and peroxisomal fractions (unpublished results), the activity of Mn-SOD being higher in the former organelles.

The confusion generated in recent years about the subcellular location of SODs in chloroplasts might be due, to a great extent, to the existence in these organelles of different compounds able to react with the O 2. - radicals generated enzymatically in the course of the SOD assays in solution, and thus showing nonspecific SOD activity. Though interfering products have reaction rate constants with superoxide radicals much lower than that of SOD (Asada et al., 1977), if their concentration is very high then their reaction with the O 2.- radicals could become significant (Halliwell et aI., 1981). On the contrary, the determination of SOD isozyme activity by acrylamide gel electrophoresis, followed by photochemical activity staining and gel densitometry, is more reliable, not only because the reactivity of soluble SODs in gels is proportional to the enzyme concentration (Bohnenkamp and Weser, 1975) but mainly because most of the compounds mimicking SOD are eliminated during the electrophoretic process. Should the first reports dealing with Mn-SOD activity in chloroplasts have presented SOD activity-stained electrophoretogram evidence of chloroplasts, the situation in this field would be much clearer today.

Some of the products present in chloroplasts known to react with superoxide radicals include: i) reductants such as ascorbate and glutathione (Asada et al., 1977; Halliwell et al., 1981); ii) free Mn(II) ions which are abundant in chloroplasts (Lumsden and Hall, 1975; Kono et al., 1976); iii) low molecular weight metal chelates, like Mnpyrophosphate (Kono et al., 1976); iv) photosynthetic electron transport components, such as cytochrome f, plastocyanin, and ferredoxin (Asada et al., 1977); and v) high molecular weight Mn-protein - pigment complexes or Mn-peptides derived there - from related to photosystem II (Foyer and Hall, 1979; Foyer and Hall, 1980). In addition, molecules with a high number of metal-thiolate clusters could also be a


Mn superoxide dis mutase and chloroplasts 435

target for O 2'- radicals, and therefore show SOD-like activity (Thornalley and Vasak, 1985).

Obviously, not all of these compounds are enzymic superoxide dismutases. The molecular properties and characteristics of these metalloenzymes have been analyzed in several review articles published in the last few years (McCord, 1979; Parker et al., 1984; Steinman, 1982) and they have been found to possess general common features though to a certain extent variable, depending upon the source of the enzyme. Apart from the molecular weight and subunit structure, important characteristics to identify a certain protein as a superoxide dismutase are the metal-protein stoichiometry, amino acid composition, specific activity, and sensitivity to inhibitors, but no doubt the most important property is the rate constant of the protein for the disproportionation of superoxide free radicals which should be of a similar order of magnitude to those determined for SODs from different sources (ca. 2 x 109 M-' sec') (Fridavich, 1983; Sevilla et al., 1982; Steinman, 1982).

Therefore, special caution should be exercised before identifying a certain protein as a superoxide dismutase on the mere basis of its SOD activity, a property relatively nonspecific and shared by many compounds, as we have mentioned above. In this respect, the recently reported characterization of a thylakoid-bound Mn-SOD from spinach chloroplasts (Hayakawa et al., 1985) could be one of these cases. The molecular weight of this protein (52,000) is considerably lower than that of Mn-SOD from pea leaves, the only SOD of this family fully characterized so far in higher plants which is 94,500 (Sevilla et al., 1982), and is also lower than the molecular weight of the partially characterized Mn-SOD from maize kernels (90,000) (Baum and Scandalios, 1981). Actually, the molecular weight reported for the Mn-SOD from spinach chloroplasts (Hayakawa et al., 1985) is also different from those described for MnSODs from mammals, fungi and bacteria (Steinman, 1982; Parker et al., 1984), and the same was true for the subunit molecular weight of thylakoid-bound Mn-SOD (Steinman, 1982). On the other hand, the Mn content reported for this new Mn-SOD is unusually low and nearly borders the trace level (0.2 atoms per molecule) in comparison with those metal-protein stoichiometries reported for Mn-SODs from different sources (1-4 atoms Mn per molecule) (McCord, 1979; Steinman, 1982; Parker et al., 1984), as well as its specific activity which is about five times lower, in terms of McCord and Fridovich's units (McCord and Fridovich, 1969). In the purification of the Mn-SOD from pea leaves, an equivalent purification procedure was used, and the possibility of a Mn loss by the enzyme molecule during the isolation procedure was ruled out by the results from the enzymatic activity reconstitution experiments. These showed that the enzyme reconstituted with Mn{II) and the native enzyme both had the same manganese content (Sevilla et al., 1980).

Therefore, the thylakoid-bound Mn-SOD recently characterized from spinach chloroplasts (Hayakawa et al., 1985), according to its molecular properties known so far, does not appear to be a characteristic Mn-containing superoxide dismutase but a protein with SOD-like activity. However, its location within the thylakoid membranes suggest that this protein could have a role as a secondary protective agent against the production of O2, - in the chloroplast, but acting in the internal structure



of the thylakoid in a similar way to that proposed by Foyer and Hall (1980) for the Mn bound to the LHC alb protein complex and other high molecular weight Mnprotein complexes isolated from photosynthetic organisms and possibly originated from photosystem II (Foyer and Hall, 1980), all of them having Mn-SOD-like activity.

Acknowledgements

J. M. P. acknowledges receipt of a research fellowship from the Caja General de Ahorros de Granada and CSIC. We are grateful to Dr. P. A. Siegenthaler and Dr. J. P. Schwitzguebel (Universite de Neuchatel, Switzerland) for their valuable suggestions on Percoll density-gradient centrifugation, and to Dr. J. J. Lazaro (Estaci6n Experimental del Zaidfn, Granada) for his assistance in the determination of chloroplast intactness. This work was supported by grant No. 603/275 from the CAICYT-CSIC (Spain).

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Manganese Superoxide Dismutase and Higher Plant Chloroplasts: A Reappraisal of a Controverted Cellular Localization