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Subcellular Distribution of Superoxide Dismutases in Rat Liver:

Cu,Zn-SOD in Mitochondria *

Running Title: Mitochondrial Cu,Zn-SOD

Ayako Okado-Matsumoto and Irwin Fridovich§

Department of Biochemistry, Duke University Medical Center, Durham, NC 27710

§ To whom correspondence should be addressed.

Tel: 919-684-5122

Fax: 919-684-8885

e-mail: fridovich@biochem.duke.edu

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 15, 2001 as Manuscript M105395200 by guest on M

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 2

ABSTRACT

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and

then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed

for both Cu,Zn-SOD and Mn-SOD by exploiting the cyanide-sensitivity of the former activity and

by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained

Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD

was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane.

The Mn-SOD associated with the inner membrane was solubilized by 0.5 M NaCl. Surprisingly

the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be

solubilized with a detergent, and to lesser degree with 0.5 M NaCl. Both the cytosolic and

mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass,

cyanide-sensitivity, SDS-sensitivity, heat stability, and chloroform + ethanol stability. Tissue

from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic

and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of

this distribution of the SODs is discussed.

Keywords: Mitochondrial superoxide dismutases / Mn-SOD and Cu,Zn-SOD in mitochondria /

Localization of SODs / SODs in organelles / SOD in lysosomes / SODs in liver /

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 3

INTRODUCTION

A small fraction of total biological reduction of oxygen occurs by a univalent pathway.

Superoxide, the first intermediate encountered on this pathway, is capable of: initiating free radical

chain oxidations; inactivating specific enzymes; and leading to the production of more powerful

oxidants by liberating Fe (II) from the [4Fe-4S] clusters of dehydratases and by reacting with nitric

oxide (1). The damaging potential of superoxide is muted by SODs, that catalyze its dismutation

to oxygen plus hydrogen peroxide; as well as by superoxide reductases (SORs), that catalyze the

reduction of superoxide to hydrogen peroxide (2).

When the subcellular distribution of SOD activity was first explored by fractionation of

liver, the cytosol was found to contain a Cu,Zn-SOD and the mitochondrial matrix a Mn-SOD. At

that time Cu,Zn-SOD was also noted in the intermembrane space of mitochondria, and in nuclei (3,

4). The cytosolic localization of Cu,Zn-SOD and the mitochondrial location of Mn-SOD was

subsequently verified by means of colloidal gold immuno cytochemistry and electron microscopy

(5, 6). However the presence of Cu,Zn-SOD in the intermembrane space of mitochondria was

called into question and attributed to contamination of mitochondrial fractions with lysosomes and

the presence of Cu,Zn-SOD in lysosomes (7).

We now report a painstaking reinvestigation of the distribution of SODs in rat liver and find

that there is indeed a Cu,Zn-SOD in the intermembrane space of mitochondria that it is the same

gene product as the cytosolic Cu,Zn-SOD. Culotta and her associates independently, and in

parallel, investigated the situation in yeast and find as we do†. We also find the Cu,Zn-SOD

strongly associated with intracellular reticular membranes isolated as microsomes.

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 4

MATERIALS AND METHODS

Material

Nycodenz was from Invitrogen Life Technologies. Xanthine oxidase was from Roche

Diagnostics Corporation. Rabbit anti-Mn-SOD antibody, sheep anti-Cu,Zn-SOD antibody, HRP

conjugated anti-rabbit IgG, and HRP conjugated anti-sheep IgG were from Upstate Biotechnology.

SP-sepharose, nitrocellulose membranes and the ECL kit were from Amersham Pharmacia Biotech.

Cytochrome c was from Fluka Chemika, while AcA54, xanthine, NAD, L-lactic acid, L-malate, uric

acid, p-nitrophenyl phosphate, benzylamine hydrochloride, and Lubrol PX were from Sigma.

Broad range prestained SDS-PAGE standards were from Bio-Rad.

Fractionation of Liver

Sprague-Dawley rats were sacrificed and the livers were promptly removed and placed in

ice cold homogenization buffer (200 mM mannitol, 50 mM sucrose, 1 mM EDTA, and 10 mM

Tris-HCl) at pH 7.4. After mincing with scissors and washing to remove blood, the liver was

homogenized in a Potter-Elvejhem homogenizer with a teflon piston, using 10 ml of the

homogenization buffer per 2.5 mg of tissue. Centrifugation at successively higher speeds yielded

the following fractions: crude nuclear fraction at 1,000 x g for 10 min; heavy mitochondria at 3,000

x g for 10 min; light mitochondria at 20,000 x g for 20 min; and microsomes at 144,000 x g for 90

min. The final supernatant was the cytosolic fraction. Each successive pellet was thrice washed

with the homogenization buffer. The centrifuges used were the Avanti J-25 centrifuge and the

Optima XL-100K ultracentrifuge, both from Beckman.

Nycodenz Density Gradient Fractionation

The procedures recommended by Nycomed Pharma and Invitrogen Life Technologies were

followed (www.nycomed-diagnostics.com and www.invitrogen.com). Nycodenz was dissolved to

50 % (w/v) in buffer containing 5 mM Tris-HCl and 1 mM EDTA at pH 7.4. This stock solution

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 5

was diluted with buffer containing 0.25 M sucrose, 5 mM Tris-HCl and 1 mM EDTA at pH 7.4.

The nuclear pellet was suspended in 25 % Nycodenz and 7.5 ml was then overlaid onto the

following discontinuous Nycodenz gradient: 3 ml 50 %, 3 ml 40 %, 4.5 ml 34 %, and 6 ml 30 %.

The sample on this gradient was topped off with 6 ml 20 % Nycodenz before sealing the tube.

The heavy and light mitochondrial pellets suspended in 10 ml of 25 % Nycodenz were placed on

the following discontinuous Nycodenz gradients: 4 ml 34 % and 7 ml 30 % and this was topped off

with 7 ml 23 % and finally 2 ml 20 %. The sealed tubes were centrifuged for 90 min at 52,000 x g

at 4 ˚C. The bands of particles seen after centrifugation have been identified by Nycomed Pharma

and Invitrogen life technologies as follows: nuclei at the 40/50 % interface; peroxisomes at the

34/40 % interface; mitochondria at the 25/30 % interface, lysosomes at the 15/20 % interface, and

Golgi membranes at the 10/15 % interface.

Enzyme Assays

SOD activity was assayed by the xanthine oxidase / cytochrome c method (8). Mn-SOD

was distinguished from Cu,Zn-SOD by assaying in the presence of 50 µM and then 2 mM NaCN

(9). Under our conditions the Cu,Zn-SOD was 24 % inhibited by 50 µM cyanide and 93 %

inhibited at 2 mM cyanide. Mn-SOD was not inhibited at these levels of cyanide. Hemoglobin,

as an erythrocyte marker, was measured by absorbance at 409 nm (7); acid phosphatase, as a

lysosomal marker, by the hydrolysis of p-nitrophenyl phosphate (10); urate oxidase, as a

peroxisomal marker, at 290 nm (11); lactate dehydrogenase (LDH), as a cytosolic marker, at 340

nm (12); fumarase, as a mitochondrial matrix marker, at 250 nm (13); cytochrome c oxidase, as a

mitochondrial inner membrane marker, at 550 nm (14); sulfite oxidase, as a mitochondrial

intermembrane space marker, at 550 nm (15); and monoamine oxidase, as a mitochondrial outer

membrane marker, by the oxidation of benzylamine (16). All assays were repeated 5 times. All

membrane fractions and samples for cytochrome c oxidase assay were treated with 0.1 % Lubrol

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 6

for enzyme assays.

Immuno Assays

Samples were subjected to 15 % SDS-PAGE, and were then blotted onto nitrocellulose

membranes under semi-dry conditions using the Bio-Rad Trans-Blot. After blocking by soaking

in 5 % dry milk proteins in 10 mM Tris-HCl, 0.15 M NaCl, and 0.05 % Tween 20, at pH 7.4 and

room temperature for 1h; the membranes were soaked with 1:500 sheep anti Cu,Zn-SOD or 1:1000

rabbit anti Mn-SOD for 1 h at room temperature. The membranes were then washed and soaked

with HRP-conjugated anti sheep IgG (1:2000) or with HRP-conjugated anti rabbit IgG (1:5000) for

40 min at room temperature. The membranes were finally washed four times and then the HRP-

containing bands were detected by luminol chemiluminescence with the ECL kit.

Submitochondrial Fractionation

Mitochondria isolated on Nycodenz gradients were separated into outer membrane,

intermembrane space, inner membrane, and matrix fractions according to Greenawalt (17) with the

following modifications: the mitoplast fraction was subjected to 3 cycles of freezing and thawing

and was then centrifuged at 144,000 x g for 90 min. The membrane fractions were washed 3

times with PBS to remove soluble proteins derived from the matrix or intermembrane spaces.

Isolation of Cu,Zn-SODs from Cytosol and Mitochondria

The procedure developed for isolation of Cu,Zn-SOD from bovine heart (18) was appliable,

with minor modifications. Thus Ion exchange chromatography was performed on SP-sepharose

and gel filtration on AcA54. The protein content of purified Cu,Zn-SOD was determined by the

Biuret method (19) and protein mixtures were assayed by the Bradford method (20) using the Bio-

Rad reagent. When assaying liver from Cu,Zn-SOD knockout mice, the tissue extracts were

purified through the acetone precipitation step before being assayed. This was done with and

without an internal standard of rat liver mitochondrial Cu,Zn-SOD to ensure our ability to recover

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 7

small amounts of activity.

Molecular Weight and Mass

Native molecular weights were determined by sedimentation equilibrium centrifugation in a

model XL-A Beckman analytical ultracentrifuge as previously described (21). Mass Spectrometry

measurements were made on a Micromass Quattro LC (Altrincham, UK) triple quadrupole mass

spectrometer equipped with a pneumatically assisted electrostatic ion source operating at

atmospheric pressure and in a positive ion mode. Apo-protein SOD samples in 50 % aqueous

acetonitrile containing 1 % formic acid were analyzed by loop injection into a stream of 50 %

aqueous acetonitrile flowing at 10 µl/ min. Native SOD samples were electrosprayed in 0.01 M

ammoniun acetate. Spectra were acquired in the multi-channel analyzer (MCA) mode from m/z

600-1400 (scan time 5 sec). The mass scale was calibrated using the multiply charged envelope of

myoglobin (MW 16951.48 Da). The raw mass spectra were transformed to a molecular mass

scale using a maximum entropy based method (Max Ent) which uses the MemSys5 program (Max

Ent Solutions Ltd., Cambridge UK) and is part of the Micromass Mass Lynx software suite.

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 8

RESULTS

SOD Activities in Liver Fractions

When rat liver was homogenized in isotonic buffer and fractionated, as described in materials

and methods, the soluble cytosolic fraction (144,000 x g supernatant) contained abundant Cu,Zn-

SOD and no detectable Mn-SOD while the heavy and the light mitochondrial fractions contained

both SODs with Mn-SOD predominating. Surprisingly, the microsomal fraction contained Cu,Zn-

SOD. Lactate dehydrogenase was followed as a cytosol marker and cytochrome c oxidase as a

mitochondrial marker. Lactate dehydrogenase was found mostly in the cytosol, with a small

amount in the microsomes; while cytochrome c oxidase was found only in the mitochondrial

fractions. These results, based on both activity assays and on immunoassays, are shown in figures

1A and 1B.

SOD and Marker Assays of Nycodenz Fractions

The particulate fractions, obtained by differential centrifugation of the liver homogenates,

were subfractionated in Nycodenz gradients and assayed for the SODs and the various marker

proteins, as described in materials and methods. The data in figure 2A demonstrate that: the

nuclear fraction was contaminated with erythrocytes and some lysosomes; the heavy mitochondrial

fraction contained some erythrocytes and lysosomes at the 20/23 % and at the 23/25 % Nycodenz

boundaries; but not at the 25/30 % boundary. The 30/34 % boundary of the heavy mitochondria

showed contamination with peroxisomes. The light mitochondrial fraction contained lysosomes at

the 20/23 and at the 23/25 % boundary and both lysosomes and peroxisomes at the 25/30 %

boundary; while the 30/34 % boundary was almost all peroxisomes.

Selected bands of particles from Nycodenz density gradients, identified by arrows in figure

2A, were assayed for SOD activity using cyanide to distinguish Cu,Zn-SOD from Mn-SOD and

this data is shown in figure 2B. The heavy mitochondrial fraction 25/30, that had been found free

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 9

of erythrocyte, lysosomal and peroxisomal markers, contained both Cu,Zn-SOD and Mn-SOD in

the activity ratio 1:5. The light mitochondrial fraction 20/23, that had been found to contain

abundant lysosomal marker activity, was rich in Cu,Zn-SOD but contained only traces of Mn-SOD.

The light mitochondrial fractions 25/30 and 30/34 contained progressively less Cu,Zn-SOD and

small amounts of Mn-SOD.

The identities of the SODs were further probed immunochemically by immunoblotting and

these results are shown in figure 2C. In agreement with the results of activity assays using

cyanide the heavy mitochondrial fraction 25/30 contained abundant Mn-SOD and much less of

Cu,Zn-SOD. This is the fraction which was free of lysosomal and peroxisomal markers and is

therefore taken to be pure mitochondria. The light mitochondrial fraction 25/30 contained both

SODs, with Cu,Zn-SOD predominant, in keeping with its content of lysosomal marker activity.

The light mitochondrial fraction 30/34 exhibited a trace of Cu,Zn-SOD and Mn-SOD.

Submitochondrial Fractions

The heavy mitochondrial fraction 25/30, which was found free of non-mitochondrial markers,

was subfractionated and assayed for several marker activities and for SODs. Figure 3A illustrates

the result of the marker assays. The matrix fraction contained primarily the matrix marker

fumarase, while the inner membrane fraction contained primarily cytochrome c oxidase, as

expected, but also a substantial amount of the outer membrane marker monoamine oxidase. The

intermembrane space contained abundant sulfite oxidase and was practically free of other markers

and the outer membrane fraction showed mostly monoamine oxidase. Immunoblotting

demonstrated that the intermembrane space contained Cu,Zn-SOD, but not Mn-SOD; while the

matrix and the inner membrane contained Mn-SOD, but no Cu,Zn-SOD. The Mn-SOD that was

associated with the inner membrane persisted despite three washings with PBS. After incubation

with PBS, PBS + 0.5 M NaCl, or PBS + 0.1 % Lubrol for 30 min on ice, followed by centrifugation

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 10

at 144,000 x g for 30 min, the 0.5 M NaCl and 0.1 % Lubrol supernatants from the inner membrane

showed about 3 times and 8 times higher Mn-SOD activity respectively than did the PBS

supernatant (Data not shown).

Isolation of Cu,Zn-SOD

The Cu,Zn-SOD was isolated from both the cytosolic and the mitochondrial fractions of rat

liver. The heavy mitochondrial Nycodenz fraction 25/30 was used, since it was demonstrably free

of non-mitocondrial markers, and the 144,000 x g supernatant fraction was taken to be cytosol.

The results of these isolation procedures are given in tables IA and IB and in figure 4, the tables

record a 45-fold purification of the Cu,Zn-SOD from cytosol and a 1,556-fold purification of

Cu,Zn-SOD from mitochondria. We can thus estimate that Cu,Zn-SOD constitutes ~2 % of the

total protein of the cytosolic fraction and only ~0.06 % of the total protein of mitochondria. Since

only ~6 % of total liver mitochondrial protein is in the intermembrane space (22) we can correct the

estimated 0.06% by multiplying it by 16.7 to get ~1 %, thus the concentration of Cu,Zn-SOD in the

intermembrane space (~1 %) is not very different from its concentration in the cytosol (2 %). We

can also estimate that mitochondrial Cu,Zn-SOD constitutes ~2.8 % of the total Cu,Zn-SOD from

table I. It should be noted that the specific activities of the Cu,Zn-SOD was ~4,700 U/mg whether

isolated from cytosol or mitochondria. Figure 4 shows that the mobility on 15 % SDS-PAGE was

identical for the Cu,Zn-SODs from both sources.

Cyanide Inhibition and Thermal Stability

The Cu,Zn-SODs derived from cytosolic and mitochondrial fractions were: isolatable by the

same procedure; of identical specific activities; and indistinguishable on SDS-PAGE.

Nevertheless it remained possible that they might differ in subtle ways that could be detected in

terms of sensitivity to inhibition by the superoxide analogue cyanide, or in terms of thermal

stability. The data in figure 5 demonstrate that Cu,Zn-SODs isolated from the cytosolic fraction,

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 11

or from the pure mitochondrial fraction, are indistinguishable in their sensitivities to cyanide

inhibition. Thus the concentration of NaCN needed for 50 % inhibition of cytosolic and

mitochondrial Cu,Zn-SODs was 230 µM in both cases. Figure 6A and 6B make the same point

with regard to inactivation at 70 ˚C and 80 ˚C. Cu,Zn-SOD is stable to SDS but Mn-SOD is not

(9). We examined the SDS stability of cytosolic and mitochondrial Cu,Zn-SODs and both were

stable for 24 h under the conditions of Geller and Winge (9) (Data not shown).

Molecular Weights and Mass

The native molecular weight of the Cu,Zn-SODs isolated from the cytosolic and the

mitochondrial fractions were determined by analytical sedimentation equilibrium. They were

found to be identical to within the limits of precision of the method ie. 31,800 and 31,400 Daltons.

The Mass of the subunits were found by electrospray mass spectrometry to be 15,820 and 15,818

Daltons respectively. These results are given in Table II.

Cu,Zn-SOD: One Gene Product

The identical properties of the Cu,Zn-SODs isolated from cytosol and mitochondria suggested

that they might be the product of one gene. This was established by examining liver from normal

control mice, transgenic mice that over express Cu,Zn-SOD, and knockout mice that do not express

Cu,Zn-SOD. The results in figure 7 show that the over expressing mice (OE1 and OE2) had 2.5

times more Cu,Zn-SOD than the controls (CT1 and CT2); while the knockout mice (KO1 and KO2)

had no detectable Cu,Zn-SOD. Since the mitochondrial Cu,Zn-SOD accounted for only ~2.8 % of

the total Cu,Zn-SOD, we processed the extract of knockout mouse livers through the acetone

precipitation step of the purification procedure (Table I), prior to assaying then for Cu,Zn-SOD

activity. To ensure the validity of this procedure one aliquot of each liver extract was doped with

an internal standard of rat liver mitochondrial Cu,Zn-SOD, added to 1.7 U/mg protein. This is the

amount of Cu,Zn-SOD derived from mitochondria in wild type mouse liver extract. After the

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 12

partial purification, no Cu,Zn-SOD activity was found in the fractions from the KO1 and KO2

mouse livers; while the aliquots that had been doped with Cu,Zn-SOD yielded fractions containing

50 U/mg of Cu,Zn-SOD activity. This procedure would have easily detected SOD in the knockout

extracts, had it been present at even 0.5 % the amount expected to have derived from mitochondria.

All expressed the same level of Mn-SOD. It thus appears that all the Cu,Zn-SOD in murine liver,

and by extension in rat liver, is the product of one gene.

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 13

DISCUSSION

Fractionation of liver homogenate by differential sedimentation into nuclear, heavy and light

mitochondria, microsomal and soluble fractions, followed by subfractionation on Nycodenz

gradients; yielded fractions that were, on the basis of marker activities, pure mitochondria and

nearly pure peroxisomes and lysosomes. The mitochondria contained both Mn-SOD and Cu,Zn-

SOD, the lysosomes only Cu,Zn-SOD, and the peroxisomes traces of both SODs, the cytosolic

fractions contained only Cu,Zn-SOD. Further fractionation of the pure mitochondria

demonstrated that the Cu,Zn-SOD was in the intermembrane space while the Mn-SOD was both in

the matrix and associated with the inner membrane. The presence of Mn-SOD in the inner

membrane fraction and of Cu,Zn-SOD in the microsomal fraction, that could be solubilized with a

detergent and also with 0.5 M NaCl, is reminiscent of the finding of lactate and malate

dehydrogenases associated with chicken liver microsomes (23). Solubilization with detergent

could signify enclosure of the SOD in membrane vesicles, but solubilization by NaCl indicates

binding to the surface of the membranes by electrostatic forces. Since phospholipids impart a

negative charge to biological membranes; cations, including protons, will be concentrated adjacent

to these membranes. Hence the pH adjacent to the membranes will be lower than in the bulk

solution and superoxide approaching the membranes would be more protonated than superoxide in

the bulk. Since hydroperoxyl radical (HO2 ) is a stronger oxidant of unsaturated lipids than

superoxide (24) it may be important to have SOD bound to membrane to intercept incoming

superoxide.

Parallels may be usefully drawn between the localization of SOD activities in mitochondria

and in Escherichia coli; thus in the gram negative bacterium the inducible Mn-SOD associates with

DNA and is most abundant in the central portion of the cell that contains the nucleoid (25); while

the homologous and constitutive Fe-SOD is concentrated in the periphery of the cells, closest to the

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 14

inner membrane. There is, in addition, a Cu,Zn-SOD found in the periplasmic space. The

lessons to be derived from this distribution are that: there are multiple sites of superoxide

generation; that superoxide does not readily cross membranes; and that superoxide cannot be

allowed a long half life in cells and consequently must be dealt with by a SOD close to its site of

generation. When applied to mitochondria these musings lead to the following deductions:

superoxide made on the inner surface of the inner membrane will be largely dealt with by the Mn-

SOD bound to that surface; superoxide made on the outer surface of the inner membrane, and hence

entering the intermebrane space, will be scavenged by the Cu,Zn-SOD in that space; superoxide is

probably also made in the matrix space and that will be dealt with by the matrix Mn-SOD.

Mitochondria are considered to be the major source of superoxide in eukaryotic cells because

they consume most of the oxygen used by these cells and because respiring submitochondrial

particles have been shown to convert 1-2 % of the oxygen consumed into superoxide and hydrogen

peroxide. The distribution of the SODs in mitochondria make it very unlikely, however, that

superoxide made in intact mitochondria could escape to the cytosol. Thus any superoxide entering,

or made in, the intermembrane space would be dismuted by the Cu,Zn-SOD or oxidized by the

ferricytochrome c in that space.

Nishijima et al. (26) have reported on the distribution of Cu,Zn-SOD in gastric mucosa using

immuno electronmicroscopy and noted this enzyme mainly in the cytoplasm but also associated

with the outer membrane of mitochondria, and partially in the membranes of the endoplasmic

reticulum and nuclei. Since they dealt with fixed sections it is understandable that the

intermembrane Cu,Zn-SOD should have appeared to be associated with the outer mitochondrial

membrane. We also detected the Cu,Zn-SOD in the microsomal fraction. This Cu,Zn-SOD

could be solubilized with 0.5 M NaCl or more effectively by 0.1 % Lubrol, for 30 min at 0 ˚C (Data

not shown).

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 15

The question of how some Cu,Zn-SOD comes to reside in the intermembrane space of

mitochondria while most of it remains in the cytosol has been answered by Culotta and associates†.

Thus they found, in yeast, that the apoenzyme can cross the outer membrane and that it is trapped in

the intermembrane space when metallated by the copper chaperone of superoxide dismutase in that

space.

The significance of SODs bound to membranes requires further comment. Polyanionic

macromolecules or polyanionic surfaces such as biological membranes will concentrate cations

from the bulk solution. This applies to protons and the result has been calculated to be a ~2 pH

unit drop in pH immediately adjacent to the polyanionic macromolecule (27, 28). The pKa of the

hydroperoxyl radical (HO2 ) is ~4.8 (29) and HO2 is a much stronger oxidant than superoxide (30,

31). It follows that superoxide diffusing forwards the polyanionic surface of the membrane could,

if allowed to closely approach that surface, protonate to HO2 and then oxidize membrane

components, such as polyunsaturated lipids. SOD bound to the membrane could prevent this by

scavenging superoxide prior to its protonation.

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29. Bielski, B. H., Cabelli, D. E., Arudi, R. L., and Ross, A. B. (1985) J. Phys. Chem. 14, 1041-

1100

30. Benson, S. W., and Nangia, P. S. (1980) J. Am. Chem. Soc. 102, 2843-2844

31. Dix, T. A., Hess, K. M., Medina, M. A., Sullivan, R. W., Tilly, S. L., and Webb, T. L. (1996)

Biochemistry 35, 4578-4583

.

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 18

FOOTNOTES

* This work was supported in part by grants from: The Amyotrophic Lateral Sclerosis

Association; The North Carolina Biotechnology Center; and Incara Pharmaceuticals Inc.

† Culotta, V. Personal communication

1 The abbreviations used are: SOD, superoxide dismutase; HRP, horseradish peroxidase; SDS-

PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate buffered

saline.

ACKNOWLEDGMENTS

We are indebted to: Dr. Manisha Patel who supplied the tissues from control, transgenic,

and knockout mice; Dr. Dave Warner who supplied the rat livers; Dr. Harvey Sage, who performed

the analytical equilibrium ultracentrifugation; and Dr. Robert D. Stevens who did the electrospray

mass spectrometry.

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 19

FIGURE LEGENDS

Fig. 1. SOD and marker enzyme activities and immunoblots of rat liver fractions.

(A) SOD and marker enzyme activities of rat liver fractions. All enzyme activities were

determined 5 times, and the mean and standard deviations are shown. Heavy mitochondria, light

mitochondria, cytosol, and microsomes fractions were isolated as described in materials and

methods. Membrane fractions were treated with 0.1 % Lubrol prior to assay for marker enzyme

activites. We defined the activities of extract of the total liver as 100 %. (B) Immunoblots of

Cu,Zn-SOD and Mn-SOD. Each lane contained 10 µg protein. The first lane contains the

supernate (20,000 x g) of liver sonicated in PBS. Following SDS-PAGE on 15 % gels, probing

with antibodies was done as described in materials and methods.

Fig. 2. Marker assays, SOD activities, and immunoblots of Nycodenz fractions.

(A) Marker assays. Hemoglobin, acid phosphatase and urate oxidase were assayed as

markers of erythrocytes, lysosomes, and peroxisomes, respectively. All enzyme activities were

determined 5 times. The arrows denote the fractions taken for activity assays (Fig. 2B) and for

immunoblots (Fig. 2C). The numbers on the abscissa represent the Nycodenz concentration

discontinuities at which bands formed. (B) SOD activities of Nycodenz fractions. All enzyme

activities were determined 5 times, and the mean and standard deviations are shown. (C)

Immunoblots of Cu,Zn-SOD and Mn-SOD. The conditions were as in the legend of figure 1B.

Fig. 3. Marker enzymes and immunoblots of mitochondrial fractions.

(A) Marker enzyme assays. Cytochrome c oxidase, sulfite oxidase, and monoamine

oxidase were assayed as markers of inner membrane, intermembrane space, and outer membrane of

mitochondria, respectively. (B) Immunoblots of Cu,Zn-SOD and Mn-SOD. The conditions were

as in the legend of figure 1B.

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Ayako Okado-Matsumoto and Irwin Fridovich Mitochondrial Cu,Zn-SOD 20

Fig. 4. Purification of Cu,Zn-SODs followed by SDS-PAGE.

Cytosolic Cu,Zn-SOD (C) and mitochondrial Cu,Zn-SOD (M) were purified. Samples

from each purification stage were applied to and separated by 15 % SDS-PAGE and were then

stained with Coomassie brilliant blue. Crude and ethanol/chloroform fractions 20 µg/lane;

acetone fractions 10 µg/lane; and purified proteins 3 µg/lane.

Fig. 5. Inhibition of cytosolic and mitochondrial Cu,Zn-SODs by NaCN.

All enzyme activities were determined 5 times, and the means are shown.

Fig. 6. Effect of heating on cytosolic and mitochondrial Cu,Zn-SOD.

(A) Heating at 70˚C. (B) Heating at 80˚C. The purified cytosolic and mitochondrial

Cu,Zn-SOD were incubated at 70 ˚C or 80˚C in 2.5 mM potassium phosphate buffer (pH 7.4) and

samples were taken at intervals for assay of residual activity. All enzyme activities were

determined 5 times, and the mean and standard deviations are shown.

Fig. 7. SOD activities in normal, transgenic, and knockout mouse livers.

We examined two mice of each normal controls (CT), Cu,Zn-SOD over expressing (OE),

and knockout (KO) mice. We used partially purified protein of knockout mice liver for Cu,Zn-

SOD assay. Another samples were used without purification. All enzyme activities were

determined 5 times, and the mean and standard deviations are shown.

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Ayako Okado-Matsumoto and Irwin Fridovichmitochondria

Subcellular distribution of superoxide dismutases in rat liver: Cu,Zn-SOD in

published online August 15, 2001J. Biol. Chem. 

  10.1074/jbc.M105395200Access the most updated version of this article at doi:

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