Eur. J. Biochem. 91, 339-344 (1978)

Subcellular Localisation and Identification of Superoxide Dismutase in the Leaves of Higher Plants

Christopher JACKSON, Jane DENCH, Anthony L. MOORE, Barry HALLIWELL, Christine H. FOYER, and David 0. HALL

Departments of Plant Sciences and Biochemistry, University of London King's College

(Received July 17, 1978)

1. The subcellular location of superoxide dismutase in the leaves of spinach and other C3 plants has been investigated.

2. Most activity appeared to be located within chloroplasts. These organelles contain a cyanide- sensitive (copper-zinc) superoxide dismutase, most of which is located in the stroma although some is bound to the thylakoids.

3 . Intact chloroplast fractions also contain a cyanide-insensitive (manganese) superoxide dis- mutase, but this activity is located on the outside of the chloroplasts and may be adsorbed onto them during isolation.

4. Leaf mitochondrial fractions contain only a small percentage of total leaf superoxide dismutase activity, but there is more than can be accounted for by contamination with chloroplasts.

5. Mitochondria contain both a cyanide-sensitive dismutase, apparently located in the in- termembrane space, and a cyanide-insensitive activity, apparently located in the matrix.

6. The microsomal fraction contains no superoxide dismutase activity.

The enzyme superoxide dismutase is present in all aerobic cells and plays an essential role in allowing organisms to survive in the presence of 0, [1,2]. Superoxide dismutases are metalloproteins, containing iron, or manganese, or copper plus zinc as the prosthetic groups [l]. Animals and higher plants contain only copper-zinc and manganese enzymes. The activities of these two types of superoxide dismutase can easily be distinguished, since the copper-zinc enzyme is inhibited by concentrations of cyanide which do not affect the manganese enzyme [l].

Chloroplasts isolated from the leaves of higher plants contain a cyanide-sensitive (copper-zinc) super- oxide dismutase [3 - 71. Some workers have reported that chloroplasts also contain a manganese enzyme [8,9] but others have denied this [6,7]. In rat liver the copper-zinc superoxide dismutase is mainly present in the cytosol whereas the manganese enzyme is in the mitochondrial matrix [ 10,111 but in human liver some of the manganese enzyme is outside the mitochondria [30]. However, Asada et al. [3] did not detect any

Enzymes. Superoxide dismutase or superoxide : superoxide oxi- doreductase (EC 1 .I 5.1.1); cytochrome c oxidase or ferrocytochrome c: O2 oxidoreductase (EC 1.9.3.1); glycollate oxidase or glycollate: 0, oxidoreductase (EC 1 .I .3.1); malate dehydrogenase or L-malate: NAD' oxidoreductase (EC 1.1 .I .38); glyceraldehyde-3-phosphate dehydrogenase (NADP') or ~-glyceraldehyde-3-phosphate: NADP' oxidoreductase (EC 1.2.1.9).

superoxide dismutase activity in the mitochondrial fraction obtained after density-gradient centrifugation of spinach-leaf extracts, although the integrity of the mitochondria was not examined. In contrast, mit- ochondria from Jerusalem artichoke tubers contain a mangano-dismutase, apparently located in the matrix space [12].

In view of these conflicting reports, we have carried out subcellular fractionation experiments designed to determine the amount and nature of the superoxide dismutase activity present in mitochondria and chloro- plasts from green leaves. Most experiments have been carried out with spinach (Spinacia oleracea) but the work has also been extended to leaves of other C3 plants.

MATERIALS AND METHODS

Reagents and Plants

Spinach leaves (Spinacia oleracea L) were obtained from our own greenhouses and from English and French commercial sources. There was no difference in the results obtained whatever the source. Barley (Hordeurn sp.) and alfalfa (Medicago saliva) were grown in our greenhouses. Nitroblue tetrazolium and xanthine oxidase were products of BDH Chemicals Ltd

340 Superoxide Dismutase in Leaves

(London, U. K.). Xanthine oxidase was also obtained from International Enzymes Ltd (Windsor, U. K.) and phosphoglycerate kinase was purchased from Boehringer (London, W5, U. K.). All other reagents were of the highest quality commercially available.

Subcellular Fractionation

Fractionation of leaf tissues was carried out essen- tially as described by Douce et al. [13]. All operations were at 0 - 4" C. 0.5 to 0.6 kg of fresh leaves were wash- ed in cold water and deribbed. The laminar tissue was cut into small pieces and added to 2dm3 of grinding medium [13]. The mixture was ground for 2 s in a one- gallon Waring Blender and the homogenate filtered through six layers of muslin and centrifuged at 1000 x g for 15min. The resultant pellet contained cell debris, nuclei and both broken and intact chloroplasts and was taken as the crude chloroplast fraction. The supernatant was decanted and centrifuged at 12000 x g for 20 min : the supernatant from this step is referred to as the soluble fraction. The pellet, containing mit- ochondria, peroxisomes and broken chloroplasts was gently resuspended in approximately 120 ml of medium containing 0.3 M mannitol, 1 mM EDTA, 20 mM 3-(N- morpho1ino)propanesulphonic acid and 0.2 % (w/v) bovine serum albumin, all adjusted to pH 7.2 with KOH. This suspension was then centrifuged at 500 x g for 10min. The supernatant was decanted and centrifuged at 11 000 x g for 15 min. The resultant supernatant was decanted by aspirator leaving a washed mitochondria1 fraction.

Intact chloroplasts (type A in the classification of Hall 1141) were prepared by the method of Walker [15]. Microsomes were isolated from the soluble fraction by centrifugation at 78000 x g for 100 min.

Enzyme Assays

All enzyme assays were carried out at 25°C. Superoxide dismutase was assayed at pH 7.8 as de- scribed by Halliwell [16] and by Henry et al. [17]. One unit (U) of dismutase activity is that amount that produces a 50 % inhibition of formazan production under the assay conditions of Halliwell [16].

Cytochrome c oxidase was determined as described by Tolbert [18], in a medium containing 0.3 M man- nitol, 10 mM KCI, 5 mM MgCI,, 10 mM phosphate and 0.2 % (w/v) bovine serum albumin at pH 7.2. Catalase was assayed using an oxygen electrode as described by Rich et al. [19]. Glycollate oxidase was measured by following the rate of oxygen uptake in an oxygen electrode (Hansatech Ltd., Kings Lynn, Norfolk, U. K.) using a 2.5 ml reaction volume containing 4 mM EDTA, 50 mM triethanolamine-HCI buffer pH 7.2, 0.001 % (w/v) Triton X-100, 60pM KCN and 50 to 400pl of sample. The reaction was initiated by the

addition of glycollate (adjusted to pH 7.2) to give a final concentration of 12mM. Activity was expressed as pmol oxygen consumed x min - ' x mg protein - '. Glyceraldehyde-3-phosphate dehydrogenase(NADP ') was assayed in a 3ml reaction volume containing 67 mM Tris-HCI pH 7.2, 3.3 mM ATP, 10 mM MgCI,, 4mM EDTA, 130pM NADPH, 3.3pg ml-' phos- phoglycerate kinase and 1 mM dithiothreitol. 50 to 100 pl of sample was incubated in this mixture for 5 min to allow activation [25] and the reaction started by the addition of 3-phosphoglycerate to give a final con- centration of 1 mM. NADPH oxidation was measured by following the decrease in absorbance of the solution at 340nm. Activity was expressed as pmol NADPH oxidised x min-' x mg protein-'.

Determinations

Protein was measured by a modification [20] of the Folin method and chlorophyll was assayed as described by Arnon 1211. Assays of the integrity of mitochondrial membranes were carried out essentially as described by Douce et a]. [13,29] and chloroplast envelope integrity was measured by the ferricyanide method [22] or by release of glyceraldehyde-3-phosphate dehydrogenase (NADP+). Before the assay of enzyme activities, chloroplasts and mitochondria were disrupted by os- motic shock [15] unless otherwise stated.

Electrophoresis

Gels of 7.5 % final acrylamide concentration were prepared by the method of Weber et a]. [23] using a gel buffer of 0.2M Tris-HCI pH8.5. 2 to 8pl of superoxide dismutase activity, containing 5 pl propylene glycol and 5 plO.l% (w/v) bromophenol blue, were layered onto the gels and run in a buffer containing 25 mM Tris and 0.192M glycine pH8.5 at 2-3mA per tube until the marker dye reached the bottom of the gel. Superoxide dismutase migrated in the direction anode to cathode and was localised by a photochemical procedure 1241.

RESULTS

Techniques are now available for isolating chloro- plasts of high integrity from the leaves of higher plants. However, it should be realised that intact chloroplast fractions are heavily contaminated with peroxisomes, mitochondria and soluble enzymes [25]. Intact mit- ochondrial fractions with high P/O ratios and good respiratory control can be isolated [13] but again there is extensive contamination with other organelles. Table 1 shows the distribution of various enzymes during the subcellular fractionation of spinach leaf tissues, in terms of the percentage of the total activity recovered

C . Jackson et al. 34 1

Table 1. SubceNular fractionation of spinach /eaves The results of a typical fractionation are presented as the activity in the fraction under consideration as a percentage of the enzyme activity recovered in all fractions. Details of the separation techniques and enzyme assays may be found in the Materials and Methods section. Glycollate oxidase is a marker enzyme for peroxisomes, cytochrome oxidase for mitochondria, glyceraldehyde-3-phosphate dehydrogenase (NADP') for intact chloroplasts and chlorophyll for total chloroplasts [25]. Comparable results were obtained in five different fractionation experiments using different batches of leaves

~~

Fraction Activity recovered in fraction

chlorophyll superoxide cytochrome c glycollate glyceraldehyde

dehydrogenase disrnutase oxidase oxidase phosphate

% total

1000 x g pellet 52 (crude chloroplast

fraction)

(crude mitochondrial fraction)

(soluble fraction)

(washed mitochondrial fraction)

12000 x g pellet 18

12000 x g supernatant 31

11 000 x g pellet 8

28

4

68

2

14

83

4

44

1 1

29

61

10

18

0

82

0

Table 2 . Subcellular fractionation of spinach leaves: specfic activities The results of the fractionation experiment described in Table 1 are presented as the specific activity of each enzyme found in each fraction. It should be noted that comparable results were obtained in each of five different fractionations using different batches of leaves.

Fraction Enzyme specific activity

superoxide cytochrome c glycollate glyceraldehyde dismutase oxidase oxidase phosphate

deh ydrogenase

U/mg protein

Initial homogenate 1.3

12000 x g pellet 3.1

12000 x g supernatant 6.6

7.4

1000 x g pellet 4.9

11 000 x g pellet (washed mitochondrial fraction)

0.003

0.019 0.088

0 0.153

0.012

0.011 0.047

0.008

0.083

0.018

0.015 0

0.019

0

found in each fraction. Table 2 shows the actual specific activity of each fraction. It may be seen that the washed mitochondrial fraction contained significant amounts of peroxisomes and broken chloroplasts. Similar results were obtained with alfalfa and barley leaves. Such contamination must be borne in mind in attempts to localise enzymes within chloroplasts or mitochondria, although peroxisomes are known not to contain super- oxide dismutase activity [3].

Table 1 shows that the crude mitochondrial fraction contained over 80 % of the cytochrome oxidase activity originally present in the leaf extract, but only 4 % of the superoxide dismutase activity. The washed mitochon-

drial fraction, which is known to contain mitochondria of high integrity [13,26], contained over 40 % of the cytochrome oxidase activity, but only 2 % of super- oxide dismutase. It may be concluded that leaf mit- ochondria contain, at most, no more than 4 - 5 % of the superoxide dismutase activity present in the leaf. The bulk of the superoxide dismutase activity present was found in the 12000xg supernatant (Table 1). Centrifugation of this fraction at 0 - 4 "C at 78000 x g for 100 min to sediment microsomes [27] did not pellet any significant superoxide dismutase activity. Hence the enzyme either is not associated with organelles in vivo or has been released from some organelle during

342 Superoxide Dismutase in Leaves

Table 3 . Distribution of cyanide-sensitive and cyanide-insensitive superoxide dismutase activities in spinach chloroplasts Type A chloroplasts were prepared by the method of Walker [15] and washed twice in cation-free medium [28]. Assays of enzyme activity were carried out as described in the Materials and Methods section, except that reaction mixtures contained 0.33 M sorbitol (which had no effect on enzyme activities). Chloroplast suspensions were either assayed directly or disrupted by osmotic shock [15] and then assayed. The results of a typical experiment are shown. In five different experiments using different batches of leaves the enzyme activities obtained varied slightly, but the latency factors were always identical

Enzyme Activity in intact Activity in ruptured Latency factor chloroplasts chloroplasts (increase in enzyme activity detected

when the envelope is ruptured)

U/mg chlorophyll.

Glyceraldehyde-3-phosphate dehydrogenase (NADP')

Cyanide-sensitive super- oxide dismutase

Cyanide-insensitive superoxide dismutase

0.14

19

41

1.61

246

41

-fold

11.5

12.9

1 .o

homogenisation and centrifugation. A likely candidate for the latter possibility would be chloroplasts, which easily lose their envelopes and stromal contents during such procedures [25]. Indeed, Table 1 shows that the bulk of the leaf superoxide dismutase activity was distributed similarly to glyceraldehyde-3-phosphate de- hydrogenase (NADP'), which is known to be a chloroplast stromal protein [25].

Chloroplast Superoxide Dismutase

To investigate this further, intact (type A) spinach chloroplasts were prepared by rapid centrifugation [15]. They were found to contain both cyanide-sensitive and cyanide-insensitive superoxide dismutase activities. A typical preparation contained 161 U/mg chlorophyll of cyanide-sensitive dismutase, and 44 U/mg chlorophyll of cyanide-insensitive dismutase. These chloroplasts were 51 intact as determined by the ferricyanide method [22]. Washing the chloroplasts twice in cation- free medium by the method of Nakatani and Barber [28] produced a chloroplast pellet that was 73 % intact and contained 246 U/mg chlorophyll of cyanide- sensitive dismutase but only 41 U/mg chlorophyll of the cyanide-insensitive enzyme.

Before assay of enzyme activities, chloroplasts are usually disrupted by osmotic shock [15]. Table 3 shows the results obtained if this procedure was omitted and sorbitol was added to the reaction medium to keep the chloroplasts intact. Unless the envelope was ruptured, only low activities of cyanide-sensitive dismutase or glyceraldehyde-3-phosphate dehydrogenase (NADP +) could be assayed. The latency factor was comparable for both enzymes (Table 3). Since glyceraldehyde-3- phosphate dehydrogenase (NADP') is known to be a chloroplast stromal enzyme 1251 these results show that cyanide-sensitive superoxide dismutase is also located within the chloroplast. In contrast, the cyanide-

A B C D Fig. 1. Superoxide dismutase activity of washed spinach mitochondria and type A chloroplasts. Washed spinach mitochondrial fractions and type A chloroplast fractions were prepared, disrupted by osmotic shock [15] and subjected to disc gel electrophoresis as described in the Materials and Methods section. The gels were stained for superoxide dismutase activity (the achromatic zones) by a photochemical procedure [24]. (A) Chloroplasts; (B) chloroplasts plus KCN; (C) mitochondria; (D) mitochondria plus KCN. Note the small achro- matic regions at the origin of the gels (see text)

insensitive dismutase did not show latency (Table 2) which indicates that it is outside the chloroplast. It may have been adsorbed onto the outside of the chloroplast during isolation, although a true location on the outside of the envelope itself cannot be ruled out. Its presence cannot be due to mitochondrial contamination, since the mitochondrial content of twice-washed chloroplast fractions is very small and, in any case, mitochondria have little dismutase activity (Table 1).

Extracts of isolated chloroplasts were also subjected to disc gel electrophoresis and the gels stained for dismutase activity by a photochemical procedure [24]. Fig.1 shows that only a single band of activity was

C. Jackson et al. 343

Table 4. Selective disruption and enzyme release from washed spinach mitochondria A washed mitochondrial fraction was prepared as described in the Materials and Methods section. 25 p1 of the fraction (about 2 mg protein) were suspended in 5 mM phosphate buffer pH 7.2 adjusted to the required osmolarity by addition of mannitok. Enzyme assays were carried out as described by Douce et al. [29]. After 5min the suspension was assayed for succinate-cytochrome c oxidoreductase or it was spun down for 1 min in an Eppendorf centrifuge at top speed and the supernatant and pellet assayed for malate dehydrogenase and superoxide dismutase activities. The results of a typical experiment are presented but comparable results were obtained in a series of six experiments carried out on different batches of leaves

Final osmolarity of suspension medium

Enzyme activity detected

succinate-cytochrome cyanide-sensitive cyanide-insensitive malate c-oxidoreductase superoxide dismutase superoxide dismutase dehydro-

genase

mOsm/l % total

315 26 11 25 11 158 56 42 33 18 86 100 100 100 100

obtained for spinach chloroplasts, which was com- pletely cyanide-sensitive. No isoenzymes were observed in several experiments. However, a small amount of cyanide-insensitive superoxide dismutase activity was present at the origin of the gels, presumably associated with membranes and so unable to enter the gels. The significance of this is unknown, but contamination of the chloroplasts by other membranous fractions, e.g. mitochondria, cannot be ruled out.

When chloroplasts were disrupted by osmotic shock, the thylakoids which could be pelleted always contained 25 - 35 % of the superoxide dismutase ac- tivity of the original chloroplasts. This activity was only gradually removed by repeated washing with homo- genising (isotonic) medium (see the Materials and Methods section), but it could be totally removed by washing with hypotonic medium (50 mM KH,PO, buffer adjusted to pH 7.8 with KOH).

Mitochondria1 Superoxide Dismutase

Washed spinach mitochondrial fractions also con- tained some superoxide dismutase activity. Three typi- cal preparations from different batches of leaves con- tained 8.4, 7.4 and 12 U of dismutase activity per mg protein. 55-65% of this activity was insensitive to cyanide and the remainder inhibited by cyanide. Disc gel electrophoresis of the mitochondrial fractions sho- wed two bands of activity, only one of which was inhibited by cyanide (Fig. l), although there was again a small amount of cyanide-insensitive enzyme activity at the origin of the gels. The mobility of the cyanide- sensitive enzyme was identical with that of the chloro- plast enzyme, which raises the possibility that its presence in the mitochondrial fraction was due only to the thylakoids presents (Table 1). Intact chloroplast fractions prepared from the same batches of leaves contained about 4U of cy anide-sensitive dismutase/mg

protein. On disruption and washing of the thylakoids (as happens during preparation of the washed mit- ochondrial fraction), this was reduced to about 1.2U/mg protein. Hence the specific activity of cyanide-sensitive dismutase in the mi tochondrial frac- tion is considerably greater than can be accounted for by contamination with thylakoids. Also, the activity of the thylakoid enzyme is not increased by exposure to hypotonic solutions, i.e. it does not show latency (see above). However, when mitochondria were assayed in iso-osmotic medium to keep them intact, only 11 - 14 % (five experiments) of the cyanide-sensitive activity could be detected (Table 4). The remainder of the activity was not seen until the mitochondrial mem- branes were disrupted. Comparable results were ob- tained with mitochondria isolated from leaves of Chenopodium album.

By gradually reducing the osmolarity of the sur- rounding medium, it is possible to achieve selective rupture of first the outer and then the inner mitochon- drial membranes. Outer membrane breakage may be followed by measuring the activity of succinate- cytochrome c oxidoreductase, which is located on the outer side of the inner membrane, so that rupture of the outer membrane allows cytochrome c to penetrate. The release of malate dehydrogenase, which is a matrix enzyme, can be used to follow rupture of the inner membrane. The bulk of cyanide-insensitive superoxide dismutase activity was not released until the inner membrane had been ruptured, as seen by the release of malate dehydrogenase. This suggests that cyanide- insensitive dismutase is a matrix enzyme, as has been reported for animal mitochondria [lo, 11, 31, 321. The results with cyanide-sensitive dismutase were less clear- cut but seemed to resemble the pattern of release seen with succinate oxidoreductase, which is consistent with the localisation of cyanide-sensitive dismutase activity in the intermembrane space, again similar to the results

344

obtained with animal mitochondria [lo, 11, 321. The well-known instability and fragility of mitochondria isolated from green leaves [13,26] makes it difficult to obtain data of greater precision.

DISCUSSION

Leaf mitochondria contain only a small percentage of the superoxide dismutase activity present in the leaf, but its distribution within the mitochondrion appears to resemble that seen in animal tissues [lo, 11, 31, 321 and in artichoke tubers [12]. Spinach-leaf mitochondria are very fragile [13,26] and so the results are not as clear-out as those obtained using mitochondria from animals or from etiolated plant tissue [12,29]. The spinach-leaf mitochondria isolated on sucrose gra- dients by Asada et al. [3] were probably completely ruptured and had lost their dismutase activity.

Chloroplasts appear to contain the bulk of leaf dismutase activity. A crude chloroplast fraction con- taining 18 % of the glyceraldehyde-3-phosphate dehy- drogenase (NADP’) activity originally present in the leaf extract contained 28 % of the superoxide dismutase activity, which suggests that almost all the leaf dis- mutase is present in chloroplasts. Mitochondria con- tain 4- 5 % of the total activity, but peroxisomes [3] and microsomes have no significant activity. If a soluble (cytosolic) leaf enzyme is present, it can only represent a very small proportion of the total activity in the leaf. In contrast, the bulk of superoxide dismutase activity in rat liver appears to be due to a cytosolic enzyme [10,11].

Most of the chloroplast superoxide dismutase ac- tivity was cyanide-sensitive (copper-zinc enzyme) and was located inside the chloroplast. The bulk of this enzyme was released on rupturing the envelope, but some remained attached to the thylakoids, although it could be removed by washing in hypotonic solution. The cyanide-insensitive (manganese) enzyme was lo- cated outside the chloroplasts, perhaps on the outer side of the envelope, or adsorbed nonspecifically to the envelope during chloroplast isolation. We can therefore explain previous reports of cyanide-insensitive dis- mutase activity located within chloroplasts [8,9] as being due to extraneous enzyme.

This work was supported by the Rank Prize Funds. A.L.M. is a Rank Prize Funds research fellow.

C. Jackson et al. : Superoxide Dismutase in Leaves

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C. Jackson, J. Dench, A. L. Moore, C. H. Foyer, and D. 0. Hall, Department of Plant Sciences, King’s College London, 68 Half Moon Lane, London, Great Britain, SE24 9JF

B. Halliwell*, Department of Biochemistry, King’s College London, Strand, London, Great Britain, WC2R 2LS

* To whom correspondence should be addressed