Biochem. J. (1975) 150, 31-39Printed in Great Britain

Subcellular Localization of Superoxide Dismutase in Rat Liver

By CHANTAL PEETERS-JORIS, ANNE-MARIE VANDEVOORDEand PIERRE BAUDHUIN

Laboratoire de Chimie Physiologique, International Institute ofCellular and Molecular Pathology andUniversite deLouvain, UCL 7539, Avenue Hippocrate 75, B-1200 Bruxelles, Belgium

(Received 31 December 1974)

The subcellular localization of superoxide dismutase was investigated in rat liverhomogenates. Most of the superoxide dismutase activity is present in the soluble fraction(84%), the rest being associated with mitochondria. No indication for the occurrence ofsuperoxide dismutase in other subcellular structures, particularly in peroxisomes, wasfound. Mitochondrial activity is not due to adsorption, since the sedimentableactivity is essentially latent. Subfractionation of mitochondria by hypo-osmotic shockand sonication shows that half of the mitochondrial superoxide dismutase activity islocalized in the intermembrane space, the rest of the enzyme being a component of thematrix space. In non-ionic media the matrix enzyme is, however, adsorbed to the innermembrane, from which it can be desorbed by low (0.04M) concentration of KCI.Superoxide dismutase activity was found in all rat organs investigated. Maximalactivity of the enzyme is observed in liver, adrenals and kidney. In adrenals, the highestspecific activity is associated with the medulla.

Superoxide dismutase is thought to play an impor-tant role in protecting cells against the toxic effect ofthe O2- radical (McCord et al., 1971; Gregory &Fridovich, 1973) and has been well characterized in alarge variety of prokaryotic and eukaryotic cells.Studies on the subcellular localization in liver havebeen published by Weisiger & Fridovich (1973a,b)and by Rotilio et al. (1973). The occurrence ofsuperoxide dismutase in the cytosol was establishedby the two groups of workers. The observations onthe association of the liver enzyme to subcellularparticles are, however, contradictory; the presence ofsuperoxide dismutase in mitochondria was claimedby Weisiger & Fridovich (1973a,b), whereas theenzyme was found exclusively in the soluble fractionby Rotilio et al. (1973). Further, examination of theexperimental evidence for the occurrence of super-oxide dismutase in mitochondria shows that anassociation of the enzyme to particles such as lyso-somes or peroxisomes has never been excluded.For the latter type of particles, this point deservescareful consideration; these particles are involvedin H202 metabolism, and a preliminary report onthe occurrence of superoxide dismutase in perox-isomes has been published (Tyler, 1973). Thepossibility of a peroxisomal localization places alsosome doubts on the intramitochondrial localizationclaimed by Weisiger & Fridovich (1973b), sincethe behaviour of peroxisomes in the fractionationsystem used by these authors is unknown.The present study was undertaken to investigate

the possible association of superoxide dismutase

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with liver peroxisomes and also to provide an analysisof the distribution of the enzyme on a quantitativebasis, by using fractionation techniques allowingclear discrimination between mitochondria, lyso-somes and peroxisomes. A survey of the activityof superoxide dismutase in various organs from ratis also included. The findings reported here have beenpublished in an abstract form (Peeters-Joris et al.,1973).

ExperimentalFractionation of homogenates by differential centri-fugation

The procedure described by de Duve et al. (1955)was followed for preparation ofhomogenates and forisolation of fractions by differential centrifugation,except that heavy and light mitochondrial fractionswere recovered in a single fraction. Four fractionswere thus isolated from the homogenate: a nuclearfraction (N), a large-granule fraction (ML), a micro-somal fraction (P) and a final supematant (S).

Analysis of the large-granule fractionThe automatic rotor designed by Beaufay (1966)

was used for density equilibrium. It was loaded withlOml of fraction ML, 32ml of a linear sucrosegradient and 6ml of a concentrated sucrose solution(density 1.32) as cushion. The procedure describedby Leighton et al. (1968) was followed for centri-fugation. Results are presented as histograms con-structed as described by de Duve (1967).

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C. PEETERS-JORIS, A.-M. VANDEVOORDE AND P. BAUDHUIN

Two systems were used to establish the submito-chondrial localization of superoxide dismutase. Insome experiments, mitochondria were swollen by themethod of Parsons et al. (1966). The washed pelletfrom fraction ML was resuspended in 20mM-potassium phosphate buffer, pH 7.2, which contained0.2g ofbovine serumalbumin/litre,and itwasadjustedto a volume equivalent to seven times the amount ofliver from which particles were isolated. This pre-paration was then analysed by density equilibrationor was used for determination ofsoluble activity.

In other experiments, a large-granule fraction in0.25M-sucrose was diluted fivefold with water, or insome cases with a solution of bovine serum albumin(20g/litre). The preparation was centrifuged at3000000g-min (rotor 40, Spinco centrifuge), yieldinga supematant (IS) which contained the constituentsof the intermembrane space. The pellet was resus-pended in water, sonicated for 15s at 90W in aBranson Sonifier (type D-12, equipped with amicrotip), and centrifuged at 3000000g-min. Thesupernatant (MA) contained the soluble matrixprotein, whereas inner and outer mitochondrialmembranes were recovered in the pellet (MB).

Enzyme determinationsSuperoxide dismutase was measured by photo-

chemical reduction of riboflavin as O2%-generatingsystem and inhibition of Nitro Blue Tetrazoliumreduction to assay the enzyme (Beauchamp &Fridovich, 1971). The incubation medium containedin a final volume of 3ml: 50mM-potassium phosphatebuffer, pH7.8; 45mM-methionine; 5.3 uM-riboflavin;84puM-Nitro Blue Tetrazolium (Sigma Chemical Co.,St. Louis, Mo., U.S.A.); 204uM-KCN; 0.5g of bovineserum albumin/litre. The amount of superoxidedismutase added to this medium was kept below1 unit of enzyme to ensure sufficient accuracy.Tubes were placed in an aluminium-foil-lined box,

maintained at 25°C and equipped with two 15Wfluorescent lamps. To provide homogeneous illumi-nation, it was found necessary to place tubes on aslowly rotating (0.Srev./min) holder. ReducedNitro Blue Tetrazolium was measured spectrophoto-metrically at 600nm after 10min exposure to light.Maximum reduction was evaluated in the absence ofenzyme. Each sample was also incubated with anexcess (40units/ml) of bovine erythrocyte superoxidedismutase, purified as described by McCord &Fridovich (1969). This allowed a correction for NitroBlue Tetrazolium reduction brought by reactions notinvolving the O2- radical.Enzyme activities were calculated from the

inhibition of reduction by using a standard curveconstructed by varying the amounts of homogenate.One unit of enzyme activity is defined as the amountofenzyme giving a 50% inhibition of the reduction of

Nitro Blue Tetrazolium (Beauchamp & Fridovich,1971).Superoxide dismutase displays latency in fraction

ML. Partial inhibition of the particulate enzymewas observed in the presence of detergents such asdigitonin or Triton X-100. Maximum activity wasobtained by sonication of the preparations, beforeassay, in a Branson Sonifier (type D-12, equipped witha microtip), at 90W for 120s, in periods of 30s, withpauses to allow for dissipation of any heat produced.Cytochrome oxidase was measured as described

by Beaufay et al. (1974), except that 0.9g of Tween-80/litre (Soci6t6 G6nerale de Produits Chimiques,Brussels, Belgium) and 0.3g of Triton X-100/litre(Rohm and Haas, Philadelphia, Pa., U.S.A.) wereincluded in the reaction mixture. Units are defined asdescribed by Cooperstein & Lazarow (1951).For the determination ofglutamate dehydrogenase,

a modification of the procedure described byLeighton et al. (1968) was followed. Advantage wastaken of the finding of Frieden (1959) that ADPpromotes the association of the enzyme subunitsnecessary for maximum catalytic activity. Thefollowing procedure was adopted: 0.2ml of 2g ofTriton X-100/litre was first mixed with 0.2ml ofparticle suspension to solubilize the enzyme; 1.4mlof a mixture containing 28.6mM-glycylglycine buffer,pH7.7, 0.57mM-NaCl, 1.47mM-EDTA, lmM-ADPand 2mM-NAD+ was then added. After a preincu-bation of 5min at 0°C, the samples were heated to25°C and the reaction was started by addition of0.2ml of 0.8M-sodium glutamate. The extinctionat 340nm was recorded during a 5min period. Asixfold increase in enzyme activity over that reportedby Leighton et al. (1968) was obtained by thismethod.One unit of enzyme activity reduces I mol ofNAD+/min.The method for the determination of adenylate

kinase is a slight modification of the techniquedescribed by Schnaitman & Greenawalt (1968).The reaction mixture contained 20mM-glucose,67mM-MgCl2, 93mM-glycylglycine buffer, pH8,lmM-NADP+, 4mM-ADP, 0.6mM-KCN, 9units ofhexokinase/ml and 0.2unit of glucose 6-phosphatedehydrogenase/ml. These purifiedenzymes (analyticalgrade) were purchased from Boehringer, Darmstadt,Germany. The mixture was kept at 0°C for about 2h,to exhaust contaminating ATP, and the reaction wasstarted by the addition of 0.1 ml of suitably dilutedenzyme to 1.5ml of the mixture. As mentioned bySchnaitman & Greenawalt (1968) the enzyme is verylabile at high dilution; hence the preparation wasdiluted with a solution containing bovine serumalbumin at 16g/litre. This was found to be essentialto avoid substantial inactivation. The extinction at340nm was recorded during Smin. Enzyme activityis expressed as pmol of ATP formed/min.

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LOCALIZATION OF SUPEROXIDE DISMUTASE IN RAT LIVER

Table 1. Fractionation ofliverhomngenate by differential centrifugation

Absolute values are in mg/g of liver for protein, in units/g of liver for enzymes. They are given as the sum of the activitiesin the nuclear fraction and in the cytoplasmic extract. Distributions are expressed as percentages of the sum of recoveredactivities in all fractions. E, Cytoplasmic extract; N, nuclear fraction; ML, large-granule fraction; P, microsomal fraction;S, supernatant. Recoveries give the sum of activities in the fractions compared with the value for fractions E+N, as apercentage. Values are the means ±S.D. for three experiments.

Absolute value(mg/gorunits/g)

Fractions ... E+NProtein 190+4.0Superoxide dismutase (total) 4178 + 403Superoxide dismutase (sedimentable) 656±63Cytochrome oxidase 16.9±6.1Acid phosphatase 5.86± 0.43Catalase 44.2+ 4.2Glucose 6-phosphatase 21.6+ 3.2

Distribution (%)

N10.6+4.12.4± 0.915.3±5.711.5±7.49.1±1.78.2+ 1.815.9± 7.9

ML29.4± 5.112.6+4.680.2+ 29.379.4±11.068.3+ 3.759.0+ 11.617.0+ 2.9

p21.2+ 2.10.7+0.54.5+3.28.3± 5.512.2+2.92.8 +2.5

64.7+ 9.8

RecoveryS (/%)

38.8±7.8 98.2+5.284.3 ±6.0 108.5±1.3

0.8±1.0 88.8+20.410.4+2.0 97.8±6.830.0±11.0 91.9+3.72.4±0.5 87.7± 13.1

1.10 1.15 1.20 1.25 1.301 a I A I

40 r(a)

1.10 1.15 1.20 1.25 1.30a I. I I

(c)30 -

20 Fl0o

01

Sol

40(b) (d)

30k

20 F(e)

lop

0

1.10 1.15 1.20 1.25 1.30 1.10 1.15

Density (g/ml)1.20 1.25 1.30

Fig. 1. Isopycnic centrifugation ofa large-granulefractionfrom Triton WR-1 339-treated rats

Density-frequency-distribution histograms of enzymes and protein after density equilibration in a sucrose gradient:(a) superoxide dismutase; (b) cytochrome oxidase; (c) protein; (d) acid phosphatase; (e) catalase. A large-granulefraction, containing the particles isolated from 5 g of liver, was layered over a sucrose gradient, extending linearlyfrom 1.15 to 1.27 in density. A cushion of density 1.32 was placed below the gradient. Centrifugation was performedat 35000rev./min for 35min. The percentage of activity in fraction ML and recovery with respect to the initialhomogenate were respectively 13.3 and 93.3 for superoxide dismutase, 67.0 and 104.6 for cytochrome oxidase, 26.7 and 96.9for protein, 45.8 and 104.3 for acid phosphatase, 71.6 and 100.2 for catalase.

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-20 v

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I 20

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.Jo

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C. PEETERS-JORIS, A.-M. VANDEVOORDE AND P. BAUDHUIN

Monoamine oxidase was measured as described byBaudhuin et aL (1964); glucose 6-phosphatase andacidphosphataseas described bydeDuveetal. (1955),except that the incubation was performed over 20hfor the latter enzyme. For these three enzymes, oneunit of enzyme activity yields 1 umol of the productof reaction/min, under the conditions described bythe above authors.

Catalase was measured as described by Baudhuinet al. (1964). The unit of activity is the amount ofenzyme that decreaws the decadic logarithm of theH202 concentration by lunit/min, in a reactionvolume of 50ml.

Protein was measured by the method of Lowryet aL (1951), automated as described by Beaufay

et al. (1974). Bovine serum albumin was used asstandard.

ResultsFractionation of liver homogenatesThe distribution of superoxide dismutase in the

fractions isolated from rat liver homogenates ispresented in Table 1, together with similar data formarker enzymes. Most of the superoxide dismutaseactivity is recovered in the final supernatant, asobserved by others in rat liver (Rotilio et al., 1973)and in chicken liver (Weisiger & Fridovich, 1973a).However, the data show a bimodal distribution of theenzyme, with a second peak in the large-granule

1.05 1.10 1. 15 1.20 1.25

(a)

(b)

(e)

(c)

1.05 1.10 1.15 1.20 1.25. . . I

(d)

I I I- L I

I .05 1.10 1.15 1.20 1.25 1.05 1.10

Density (g/ml)

I . I 1.2I1.15s 1.20 1.2.5

Fig. 2. Isopycnic centrifugation ofa large-granule fraction, after swelling ofmitochondria

Density-frequency-distribution histograms of enzymes and protein after density equilibration in a sucrose gradient:(a) cytochrome oxidase; (b) glutamate dehydrogenase; (c) superoxide dismutase; (d) adenylate kinase; (e) monoamineoxidase; (f) protein. The medium of Parsons et al. (1966) was used for swelling mitochondria. The preparation, containingthe particles isolated from 1.4g of liver, was layered over a sucrose gradient extending linearly from 1.05 to 1.25 indensity. A cushion of density 1.32 was placed below the gradient. Centrifugation was performed at 3500Orev./minfor 35min. The percentage of activity in fraction ML and recovery with respect to the initial homogenate were respectively68.7 and 90.0 for cytochrome oxidase, 86.8 and 100.4 for glutamate dehydrogenase, 12.2 and 91.4 for superoxidedismutase, 45.1 and 83.4 for adenylate kinase, 63.4 and 104.8 for monoamine oxidase, 29.2 and 94.9 for protein.

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I-30 -

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50

40

30

20

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0

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Oj01

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LOCALIZATION OF SUPEROXIDE DISMUTASE IN RAT LIVER

fraction. This is clear when the distribution ofsedimentable enzyme is calculated. It is, however, notpossible with this type of experiment to know if theparticulate enzyme is associated with mitochondria,lysosomes or peroxisomes, since those particles areall concentrated in the large-granule fraction.

Fractionation of the large-granulefraction

The intracellular localization of the superoxidedismutase found in the large-granule fraction wasfurther studied by isopycnic centrifugation. Toseparate lysosomes from mitochondria and peroxi-somes, 170mg of Triton WR-1339 (Rohin and Haas)was injected intravenously to the rat, 3 days beforedeath, as described by Wattiaux et al. (1963). Densitydistributions are presented in Fig. 1.

It is clear that superoxide dismutase has a distri-bution similar to the mitochondrial marker, cyto-chrome oxidase. Adsorption of soluble enzyme tomitochondria is unlikely. Indeed, it is hardly com-patible with the latency of mitochondrial superoxidedismutase. When the enzyme from a large-granulefraction is measured in an iso-osmotic incubationmedium (0.25M-sucrose) to preserve the integrity ofparticles, it is found that the activity representsbetween 10 and 20% of the activity observed afterdisruption of granules by sonication. Further, wehave verified that addition of 0.3M-KCI to the sus-pension medium of a large-granule fraction does notsolubilize mitochondrial superoxide dismutase.The density distribution of superoxide dismutase

does not show any evidence of a peroxisomallocalization. It can be calculated from the dataof Fig. 1 that, for the two fractions corresponding tothe peak of the distribution of catalase, the latterenzyme is concentrated 13-fold with respect tosuperoxide dismutase. Taking into account theamount of these two enzymes present in the large-granule fraction and the contamination of theperoxisomal peak by mitochondria, it can be inferredthat if any superoxide dismutase is associated toperoxisomes it represents less than 1 % of the enzymeactivity of the homogenate.

Localization ofsuperoxide dismutase in mitochondria

Density distributions observed after swelling themitochondria of a large-granule fraction in themedium of Parsons et al. (1966) are presented inFig. 2. The distribution of adenylate kinase, which islocalized in the intermembrane space, shows that theexternal mitochondrial membrane was disrupted.Under the conditions used for density equilibration(35min at 35000rev./min), soluble protein does notsediment appreciably; it is clear that adenylatekinase remains essentially in the top fractions of thegradient, corresponding to the sample zone before

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centrifugation. Further, the distributions of cyto-chrome oxidase and glutamate dehydrogenase showthat the inner membrane remained fairly intact:the two enzymetg used respectively as markers for theinner membrane and the soluble matrix protein, havesimilar distributions. Most outer mitochondrialmembranes remain associated with the inner-mem-brane-matrix complexes, since the amount of mono-amine oxidase found around a density of 1.11, wherefreed outer mitochondrial membranes equilibrate,isMaLThe distribution pattern of superoxide dismutase

in Fig. 2 is bimodal, half of the enzyme remaining inthe upper fractions, like adenylate kinase, whereas therest equilibrates at a density of 1.19, like cytochromeoxidase. In view of these results, two interpretationscan be proposed: either we are dealing here with adouble localization of the enzyme in mitochondria,or the distribution observed for superoxide dismutasecan be explained by a secondary adsorption ofsoluble

0 20 40 60 80 100

KCI

Glutamate dehydrogenase

KCI

KCI

Superoxide dismutase

Adeny1.. kinmse

o20 40 60 80 lOGSoluble activity (%)

Fig. 3. Solubilization ofmitochandial,superoxide dismutaseafter disrtion of the outer mitochondrial membrane and

influence ofKCI in the suspension mediwnAfter swelling of mitochondria in the medium of Parsonsetal. (1966),0.3 m-KClwasadded to aportion ofthe particlesuspension. The two preparations, with and without KCI,were centrifuged at 3000000g-min (rotor 40, Spincocentrifuge) and the activity was measured in the super-natant. Soluble activity is expressed as a percentage of theactivity in the large-granule fraction.

35

C. PEETERS-JORIS, A.-M. VANDEVOORDE AND P. BAUDHUIN

IS MA MB IS MA MB

4

2

9!

0C)

0w

4

2

0

(a) (c)

(d)(b)

-2

1:

*;)I I24..-v._

2 o-

(e)

o I

0 20 40 60I II ..

80 100 0 20 40 60 80 100

Protein (% of total)

Fig. 4. Localization ofsuperoxide dismutase in submitochondrialfractions

(a) Superoxide dismutase; (b) adenylate kinase; (c) glutamate dehydrogenase; (d) cytochrome oxidase; (e) monoamineoxidase. Mitochondria from a large-granule fraction were fractionated, as described in the Experimental section, to separatethe intermembrane space (IS) from soluble matrix (MA) and inner and outer membranes (MB). Relative specific activity(% enzyme activity/% protein is expressed with respect to the initial large-granule fraction. Results are the average oftwo experiments.

intermembrane enzyme to mitochondrial outer orinner membranes. The second possibility deservescareful consideration, since it was observed withnucleases present within the intermembrane space ofmitochondria (Baudhuin et al., 1970).To distinguish between these two possibilities,

KC1 was added to the suspension medium, afterswelling of mitochondria. Fig. 3 shows that nodesorption of superoxide dismutase occurred inthe presence of 0.3 M-KCI. Similar results wereobtained after swelling mitochondria in 0.05M-sucrose. Further, latency of superoxide dismutasewas still observed under conditions where completesolubilization of adenylate kinase was achieved.When the mitochondrial outer membrane is ruptured,free activity of superoxide dismutase is only 52% ofthe maximum activity.

At this point, it may be concluded that superoxidedismutase is associated partly with the intermembranespace and partly to either the matrix or the inner mito-chondrial membrane. To localize the enzyme moreprecisely, mitochondria were subfractionated asdescribed in the Experimental section to separate theconstituents ofthe intermembrane space (IS), those ofthe soluble matrix protein (MA) and inner and outermembranes (MB). Asshown in Fig. 4, halfofthe mito-chondrial superoxide dismutase is found associatedwith the marker for the intermembrane space(adenylate kinase) and the rest remains in the fractionscontaining the markers for inner and outer mem-branes (cytochrome oxidaseandmonoamine oxidase).Adsorption is again a possibility, since the inner mito-chondrial membrane was intact in the desorptionattempts reported in Fig. 3. To verify this point, the

1975

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LOCALIZATION OF SUPEROXIDE DISMUTASE IN RAT LIVER

> 60

40

20

C, A A A

.5 I

0 0.05 0.10 0.15

[KCl] (M)Fig. 5. Solubilization of residual mitochondrial superoxidedismutase, after elimination of intermembrane-space

components

A large-granule fraction in 0.05M-sucrose was centri-fuged at 3000000g-min (rotor 40, Spinco centrifuge) toeliminate intermembrane protein. The pellet was resus-pended in water and sonicated. After addition of KCI invarious amounts, the suspension was centrifuged again at3 000000g-min. Soluble activity is expressed as apercentage of the activity found in the sonicated prepara-tion. 0, Glutamate dehydrogenase; *, superoxidedismutase; *, cytochrome oxidase.

experiment reported in Fig. 4 was repeated, but KCIwas added in various amounts to the particle sus-pension immediately after sonication, before the lastcentrifugation. In Fig. 5, the proportion of solubleactivity, i.e. the distribution of superoxide dismutasebetween fractions MA and MB, is given as a functionofKCI concentration. The distribution of the enzymeis changed completely by KCI; most of the enzyme isfound soluble in KCI at a concentration of 0.04Mor more. In view of the low concentration of KCInecessary to solubilize the enzyme, this component ofthe mitochondrial superoxide dismutase activity canbe considered as associated with the matrix.A double localization of mitochondrial superoxide

dismutase is thus demonstrated by our results. Thisobservation agrees with the results of Weisiger &Fridovich (1973b) on chicken liver. The enzyme acti-vity is divided roughly equally between the matrix andthe intermembrane space. We have also confirmedthe observation of Weisiger & Fridovich (1973b) thatthe matrix enzyme is not sensitive to KCN, whereasboth the cytosol enzyme and the intermembraneenzyme are inhibited by 1 mM-KCN (Fig. 6).

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[KCN] (mM)Fig. 6. Effect of cyanide on superoxide dismutase

Superoxide dismutase activities are expressed with respectto the preparations in the presence of 0.01 mM-KCN. Atthis concentration, KCN does not significantly affectthe enzyme activities. *, High-speed supernatant of thehomogenate: 0, fraction IS; A, combined fractions MAand MB.

Superoxide dismutase activity in other tissues

A survey of the superoxide dismutase activity invarious rat organs is presented in Table 2. Liver wasfound to be the richest in superoxide dismutase.Except for adipose tissue, the enzyme was found in allorgans investigated, the adrenals showing a specificactivity nearly equal to that of liver. This may berelated to a possible role of superoxide radicals inauto-oxidation of adrenaline (Misra & Fridovich,1972). In view of this observation, medulla and cortexwere separated from bovine adrenals. Both on a wet-weight basis and on a protein basis, a threefold higheractivity was found in the medulla. For four experi-ments, average activities ± S.D. were 3100± 402 units/gfor the medulla and 1095±297units/g for the cortex;corresponding values for the specific activities were28± 3.1 and 10± 3.4units/mg of protein.

Discussion

The dual localization of superoxide dismutase inthe soluble fraction and in mitochondria is clearlydemonstrated by the combination of differential andisopycnic centrifugation. Our findings are in goodagreement with the observations of Weisiger &Fridovich (1973a,b) on chicken liver, but the intra-cellular localization found here is in contradictionwith the results published by Rotilio et al. (1973)

37

38 C. PEETERS-JORIS, A.-M. VANDEVOORDE AND P. BAUDHUIN

Table 2. Superoxide dismutase activity in various organsofthe rat

In pancreas homogenates, superoxide dismutase activityis rapidly inactivated, even when the preparation is keptat 0°C. The value given was obtained in a single experimentby measuringtheactivitywithin 30min ofhomogenization.Statistics refer to averages±S.D., for three experiments.

Activity Specific activityOrgan (units/g) (units/mg of protein)

Liver 4480±1165 22.1+6.2Adrenal 1563±1053 19.7+5.0Kidney 1342± 383 12.9±3.0Blood 785± 317 3.6+1.0Spleen 441± 95 4.7+1.1Heart 421+ 109 8.6+2.0Pancreas 300 1.5Brain 262± 68 2.8±0.6Lung 200± 55 3.1+1.0Stomach 140± 30 6.5+2.2Intestine 122± 43 2.5± 1.5Ovary 112± 45 2.0+0.8Thymus 88± 42 1.3±0.5Fat 0 0

on rat liver. The latter authors give few details oftheirexperimental procedure, but it appears that theirassay system for superoxide dismutase did not takeinto account the mitochondrial enzyme. In ouropinion, the latency of the mitochondrial enzyme ismost probably responsible for this discrepancy.Unfortunately the composition of the reactionmedium for superoxide dismutase assay is not givenby Rotilio et al. (1973).With respect to latency of superoxide dismutase,

no data comparable with ours exist in the literature.However, in aerobic flagellates, where the enzyme isassociated with hydrogenosomes, Lindmark &Muller (1974) have shown that superoxide dismutaseis also latent. For particulate superoxide dismutase,latency should be most likely interpreted as a restric-tion to the penetration of02--generating system or ofNitro Blue Tetrazolium, rather than as a slow pene-tration of the O2-* radical through the mitochondrialmembrane.At variance with the observation of Tyler (1973),

no significant amount of superoxide dismutase couldbe detected in peroxisomes. It is worthwhile men-tioning here that in spinach leaves Asada et al. (1973)were also able to separate, by gradient centrifugation,particulate catalase from superoxide dismutase inlarge-granule fractions. Further, as pointed out bythese authors, in this material an association withmitochondria, in addition to the localization inchloroplasts, is not excluded by their results.By comparison with cytochrome oxidase, and

assuming that all the sedimentable superoxidedismutase is associated with mitochondria, our results

show that in rat liver the mitochondrial enzyme repre-sents 16% of the activity of the homogenate. Onaverage, superoxide dismutase activity of mito-chondria was partitioned 56% in the intermembranespace and 44% in the matrix (13 experiments, S.D. ±10%); these values correspond respectively to 9 and7% of the activity found in the homogenate. Ourcalculation does not account for the 10% inhibition ofthe soluble and intermembrane enzyme at the KCNconcentration used for the assay (see Fig. 6). A correc-tion can be introduced, but is found to be small andwithin experimental error.Thesurvey ofthe activity invarious organs confirms

the wide distribution of the enzyme, liver displayingthe highest specific activity of superoxide dismutase.Our value for superoxide dismutase in liver is in goodagreement with that of Lindmark & Muller (1974),who measured the activity in rat liver with xanthineoxidase as O2--generating system and cytochrome creduction for detection.As shown by Weisiger & Fridovich (1973b), mito-

chondrial superoxide dismutases can be clearlydifferentiated by their sensitivity to cyanide. Thecyanide-insensitive matrix enzyme is a mangano-protein, similar to the bacterial superoxide dismutase.Both intermembrane mitochondrial superoxidedismutase and cytosol enzyme have been shown to besensitive to cyanide, and they are considered byWeisiger & Fridovich (1973b) to be a cuproprotein.We thank Dr. C. de Duve for his interest in this work and

Dr. H. Beaufay for suggesting improvements in the manu-script. We are grateful to Mrs. M. J. Ledrut-Damanetand Miss N. Delflasse for their technical assistance. Thiswork was supported by grants from the Belgian FondsNational de la Recherche Scientifique (F.N.R.S.), Fondsde la Recherche Fondamentale Collective (F.R.F.C.)and Minist6re de la Politique et ProgrammationScientifique.

References

Asada, K., Urano, M. & Takahashi, M. (1973) Eur. J.Biochem. 36, 257-266

Baudhuin, P., Beaufay, H., Rahman-Li, Y., Sellinger,0. Z., Wattiaux, R., Jacques, P. & de Duve, C. (1964)Biochem. J. 92, 179-184

Baudhuin, P., Bartholeyns, J. & Peeters-Joris, C. (1970)Arch. Int. Physiol. Biochim. 78, 985-987

Beauchamp, C. & Fridovich, I. (1971) Anal. Biochem. 44,276-287

Beaufay, H. (1966) La Centrifugation en Gradient deDensite, Application d l'Etude des Organites Sub-cellulaires, pp. 1-132, Ceuterick, Louvain

Beaufay, H., Amar-Costesec, A., Feytmans, E., ThinFs-Sempoux, D., Wibo, M., Robbi, M. & Berthet, J. (1974)J. Cell Biol. 61, 188-200

Cooprstein, S. J. & Lazarow, A. (1951)J. Biol. Chem. 189,665-670

de Duve, C. (1967) in Enzyme Cytology (Roodyn, D. B.,ed.), pp. 1-26, Academic Press, New York

1975

LOCALIZATION OF SUPEROXIDE DISMUTASE IN RAT LIVER 39

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