BIOCHEMICAL

Vol. 67, No. 3, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SUPEROXIDE DISMUTASE OF BOVINE AQ'IDFROG ROD OUTER SEGMENTS MICHAEL 0 . dHALL Jules Stein Eye Institute, University of California,

The Center Los Angeles, D.O.

for the Health CA 90024.

HALL

University of London King's College, 68 Half Moon Lane, London SE24 gJF.

Received

August

10,

Sciences,

Department

of Plant

Sciences,

1975

SiTIvlMARY Bovine rod outer segments (ROS) contain soluble superoxide dismutase 1]hich from cyanide sensitivity and electrophoretic mobility appears identical to Cu-Zn SOD of erythrocytes. Enzyme activity of ROS extracts is 200-400 times as much as remainder of retina. Frog ROS also contains a cyanide-sensitive SOD which is not due to erythrocyte contamination since the retina is avascular. SOD in ROS may inhibit free radical oxidation of polyunsaturated fatty acids. In light, high oxygen concentrations in developing retina may activ:te lipid peroxidation leading to retrolental fibroplasia. High concentrations of ascorbic acid in the retina may act as a protective mechanism against superoxide. INTRODUCTION animal

Superoxide and it

systems(l),

primary

defense

univalent

results

in

inner

against

reduction

of premature

cell

retinas

products

to light

for

of mammals are well

tissue.

receive

nutrients

epithelium.

Recently

produced some years

it

of photoreceptor

oxygen

of

the rod

outer

segments

acids(j)(particularly

docosahexaenoic it

seemed likely

The

vascularized,

while

by diffusion

outer a high acid)

that

a mechanism

the

present exposure

of

oxidation

segments(2).

which

the

from

radical

level

retinas which

fibroplasia.

of free

(ROS) contain

by the

tension,

has been shown that

cell

and the

that

Thus oxykven is

in the accumulation

phase

significance(4),

to high

plant

enzyme constitutes

has been known by exposure

the pigment

results

this radical,

lipid

fatty

a functional

neural

that

in numerous

known as retrolental

segments

outer

identified

superoxide

in the

the lipids urated

condition

through this

It

are damaged

of the retinas

choriocapillaris

frog

the cytotoxic

the blinding

photoreceptor

has been

has been proposed

of oxygen.

infants

portions

throughout

dismutase

Since

of polyunsatappear should

to have be

BIOCHEMICAL

Vol. 67, No. 3, 1975

AND BIOPHYSICAL

present in the ROSto prevent free radical fatty

acids.

of these polyunsaturated

Superoxide dismutase (SOD) would provide such a protective

mechanism. In light retina

oxidation

RESEARCH COMMUNICATIONS

of this,

and the recent demonstration(5) that the entire

contains SOD, we examined the ROSof cows and frogs for the presence

of this enzyme. METHODSBovine eyes were obtained from a local slaughterhouse and were kept on ice, in the dark, until light,

until

light.

the dialysis

The anterior

poured out. buffer,

dissection,

which was carried out under dim red

step, at which point the preparation was exposed to

segment of the eye was removed and the vitreous

The eyecup was rinsed with 0.6$ NaCl in 0,04M potassium phosphate

pH 7.8 (PBS).

The retinas were dissected from the eyecup, rinsed

gently in cold PBS, and dropped into a centrifuge After

dissection

twenty times to liberate

5 min at 50g.

and the procedure was repeated.

at 2000g for 10 min.

three times by centrifugation

which remain in the supernatant (J. Heller,

The pellet

of ROSwas diluted

a glass tube with a loose fitting

The

at 50g for 10 min to remove retinal

The supernatant was centrifuged

of ROSwas washed a further

cation).

in the cold for

The supernatant, which contains the ROS, was removed, two

combined supernatants were centrifuged

erythrocytes,

cold PBS.

'The tube was capped, gently

the ROS, and centrifuged

volumes of PBS were added to the pellet

debris.

tube containing

was completed, PBSwas added to the tube so that the volume

of solution was twice the volume of the retinas. inverted

was

The precipitate

in PBS to remove personal communi-

to 5 ml with PBS and homogenized in

teflon

pestle (0.5

run clearance).

This

fragments the ROSwhile causing minimal breakage of residual erythrocytes. The ROSfragments and erythrocytes

were removed by centrifugation

for 1 hr and the supernatant was dialyzed phosphate buffer,

pH 7.8 (0.5KP7.8).

overnight against 0.05M potassium

The retinal

residue remaining after

detachment of the ROS, was homogenized in PBS in a loose fitting homogenizer. dialyzed

After

centrifuging

against 0.5ii7.8

at 55,OOOg

teflon-glass

at 55,OOOgfor 1 hr, the supernatant was

overnight.

ROSand retinal

1200

ex.tracts were prepared

BIOCHEMICAL

Vol. 67, No. 3, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

100

b

0

80-

J of photoreduction of NbT by (a) extract of bovine rod Figure 1. Inhibition outer segments and (b) extract of bovine retina. SOD activity was measured spectrophotometrically in the ROS and retinal extracts by the photoreduction of nitro blue tetrazolium (NRT) in a total volume of 1.0 ml(T). %en the extracts were boiled (x), or when 10 PI, of 0.2M KCN was added to the reaction mixture (I), no inhibition of NBT reduction occurred.

from

dark-adapted

Protein

in

of Lowry,

frog

eyes by the

the dialyzed et a1.(6),

prepared

according

in 0.44$

amido

ROS and retinal SOD activity

1-zxtracts

Protein

followed

described

for

bovine

was determined

was localized

to Liavis(8).

black,

technique

eyes. by the method

on polyacrylamide

was stained

by electrophoretic

disc

by immersing destaining

gels(T)

the gels

in

lO$ acetic

anounts

of bovine

acid. FE3JLTS

Figure

ROS extract could

inhibition

of adding

to the NBT photoreduction

system.

route is

inhibition equivalent

with

crude

with

extract

transfer lo-20

only

achieved

to 10.24

The maximal

is from

Figure

the bovine

increasing

about

retina

mg of protein.

photoreduction

was completely

released

upon addition

to the reaction

mixture

inhibition

lb shows

by boiling

an oxygen

to NUT(7).

Maximum

which

is

the inhibition In this

systems,

the

the dialyzed

maximal extract,

inhibition extract,

of 10 ~1 of 0.2M KCN buffered

1201

equiva-

of photo-

case,

800 ~1 of the retinal In both

which

indicating

ROS extract,

extract.

upon adding

68$,

riboflavin

~1 of bovine

ug of protein.

of NBT with is

this

of electron

reached

to 22.6-45.2

reduction

is

the effect

be achieved

independent

lent

la shows

which of or to pH 7.8.

Vol. 67, No. 3, 1975

BIOCHEMICAL

b

a

Figure 2. Polyacrylamide dismutases. (a) purified (c) frog ROS extract; (d) Gels were stained for SOD

A curious retinal

phenomenon

extract.

is observed inhibition reaction

mixture.

200 ~1 of retinal activity,

Dialyzed

was between

boiled

this

system.

Figure

2 shows

and frog

prior

that

added

assay.

resulted

inhibition

mobility

dismutase as pure

to electrophoresis,

frogs

from

bovine

bovine or if

which

patterns

1202

results

could

staining

SOD.

in

be achieved amounts that

of

of SOD activity bovine

of ROS some

the reduction

as purified

ROS and retina

erythrocyte the

than

not due to SOD

indicating with

as well

extracts,

is

of increasing

was interfering

gel

to the

when less

gave spurious

inhibition,

disc

is added

of NBT.

Addition

in decreasing

ROS and retinal

No further

mixture

The maximum inhibition

the polyacrylamide

of inhibition

observed

reduction from

the bovine

(5-10s)

extract.

to the reaction

of the extracts

Superoxide

amount

200 11 of extract

the

extracts

of soluble superoxide (b) bovine ROS extract; (e) frog retina extract.

when studying

retinal

30s and 40$ of the control.

component

electrophoretic

a small

to some non-specific

ROS and retinal

endogenous

SOD,

lb,

more than

is

extracts

cyte

until

extract

or retinal

bovine

observed

of dialyzed

suggests

the NBT photoreduction

NBT in

25 ul

This

but rather

e

disc gel electrophoresis bovine erythrocyte SOD; bovine retina extract; activity.

was repeatedly

can be induced

RESEARCH COMMUNICATIONS

d

C

As shown in Fig.

upon adding

AND BIOPHYSICAL

from

erythro-

has the same If

solutionsare

the extracts

are

made 2 mN

Vol. 67, No. 3, 1975

with

KCN, the

and retina.1 amide

SOD activity

extracts

black,

activity

BIOCHEMICAL

but

is

the major

always

which

be removed Since

during

erythrocytes

ROS preparation,

mined

as cyanohemiglobin,

fresh

blood

assay,

From its from bovine protein, remainder

Both

has the

with

band of SOD just

above

is

same sensitivity

the enzyme has a different

bands

gels

source

of SOD activity

not affected assay,

are

seen.

by the contami-

which

a solution

(the

must

therefore

frog

retina

erythrocytes(1).

and its

identical

ROS extracts

of the retina. retinas,

to be about

were

tested

was seen.

to the Cu-Zn

containing

No quantitative

estimate

1203

contamination.

mobility,

200 to 400 times

since

dismutase. the enzyme

SO3 isolated per microgram

from of

as much SOD as does

could

is

the

be made of the amount

by endodenous there

red

the SOD activity

superoxide

of enzyme activity

However,

2 x lo6

erythrocytes

Thus

a soluble

due to the interference

the frog

in the spectrophotometric

contain

assay.

lysed

electrophoretic

On the basis

from

By the cyanohemiglobin

When 2 x lo4

ROS contain

was deter-

and thus

was not due to erythrocyte

and frog

the

prepared

is avascular,

was calculated

of NBT photoreduction

ROS appears

of the ROS extract

contanination).

ROS extract

contaminate

of cyanohemiglobin

ROS extract)

sensitivity,

spectrophotometric

of SOD, and could

content

contamination

bovine

cyanide

bovine

stained

in some cases,

However,

No minor

per ml of ROS preparation.

of SOD in frog the

using

in the bovine

bovine

SOD.

the hemoglobin

to 10 1.11bovine

DISCUSSION

are

the ROS

ROS and retina,

on disc

has no erythrocyte

no inhibition

present

and,

Both

A minor

ROS and retina

are a rich

the erythrocyte

(equivalent

when gels

the spectrophotometric

as a standard(g)

ROS preparation

cells

abolished.

electrophoresis.

bovine

blood

frog

on the gel.

with

bands

of bovine

of SOD activity

intereferes

completely

below

and to KCN as does bovine

The localization

assay,

just

from

mobility

RESEARCH COMMUNICATIONS

band of SOD activity.

in extracts

dismutase

electrophoretic

nant

one major

is

protein

seen migrating

SOD band

to boiling

on the gels

show numerous

only

Superoxide

AND BIOPHYSICAL

substances

no contamination

with of

Vol. 67, No. 3, 1975

BIOCHEMICAL

frog ROSby erythrocytes,

AND BIOPHYSICAL

the band of activity

RESEARCH COMMUNICATIONS

seen on polyacrylsmide gels is

due solely to SODpresent in the ROS. The reason for this high concentration lative.

Free radical

oxidation

of SODin ROScan only be specu-

reactions induced by light

have been shown to

occur in frog ROS(2).

'The action spectrum of light-induced

in frog ROSis similar

to the absorption spectrum of rhodopsin,

be present to inhibit cant polyunsaturated vertebrate

this free radical fatty

ROSstudied.

in the lipoprotein

peroxidation Thus SODmay

of the functionally

signifi-

acids which are abundant in the phospholipids of all It

has been suggested(lO,ll)

membranesof the developing retina

high oxygen concentration, obliterative

oxidation

lipid

and light,

phase of retrolental

may be sufficient

fibroylasia.

ascorbic acid content of the retinas

that lipid

peroxidation

in the presence of a to start

Of interest

the vaso-

is the high

of a number of mammals(l2). Reduced

ascorbate can react with the superoxide radical(l3,14), can subsequently be decomposedby catalase;

producing HsOs, which

this enzyme has not been demonstr-

ated in ROS, but is present in microperoxisomes in the adjacent retinal pigment epithelium(l5). Acknowledgements We thank Ms. G. Otova for technical assistance. This work was supported by a grant from the National Institutes of Health to M.O.H. REFERENCES 1. 2. :. 5* 6. 7. 9": 10. 11. 12. 13. 14. 15.

Fridovich, I. (1974) Advances in Enzymology, 4l, 35-96. Kagan, V.E., Shvedova, A.A., Novikoff, K.N. and Koslov, Yu.P. (1973) Biochim. Biophys. Acts., 3 0, 76-79. Anderson, R.E. and Maude, 1!1.B. 3 1970) Biochemistry, 2, 3624-3628. ijheeler, T.G., Benolken, R.M. and Anderson, R.E. (1975) Science, 188, 1312-1314. Fried, R. and Mandel, 't'. (1375) J. Neurochem., 2, 433-438. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem.1pz, 265-275. Beauchamp, C. and Fridovich, I. (1971) Anal. Biochem., a, 276-287. Davis, B.J. (1964) Ann. N.Y. Acad. Sci., 121, 404-427. van Assendelft, 0.W. (1970) Spectrophotometry of Haemoglobin derivatives. Royal Van GorcumLtd., Assen, The Netherlands. Riley, P.A. and Slater, T.F. (1969 L+ncet, G. 265. Slater, T.F. and Riley, P.A. (19701 Lancet, a, 467. Heath, N., Beck, T.C. and Rutter, L.C. 11961). Vision Res., _1. 274-286. tipel, B.L. and Neumann,J. (1973) Biochim. Xophys. Acta, m, 520-529. Allen, J.F. and Hall, D.O. (1973) Biochem. Biophys. Res. Commun.,z, 856-862. Leuenberger, P.F., and Novikoff, A.3. (1975) J. Cell Biol., s, 324-334. 1204