Vol. 81, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 469-472

March 30,1978

ACTIVE

SUBUNITS

Joe V. Bannister, Dept.

FROM

Angela

of Physiology

Anastasi

February

DISM UTASE

and William

and Biochemistry, Msida

Received

SUPEROXIDE

University

2,1978

When copper-zinc

superoxide

dismutase

is dialyzed

against

the enzyme

dissociates

8 M urea,

are catalytically

Introduction

enzyme

contains

2 atoms

functional

metal

with

a molecular

be obtained

in the presence

bridges

however,

reported

into monomers

from

swordfish

urea

(4).

erythrocyte decided

of about

16,000

that during dissociates

For-man

enzyme to investigate

is active

lauryl

could

and Fridovich wheat

from

(3)

germ

sulphate

have,

dissociates

alone,

whilst

the enzyme

in the presence

had reported

of urea

only

even though

electrophoresis,

in the presence

the effect

to

that no interchain

into monomers

and Fridovich

is the

erythrocytes

weight

from

detergent-gel

and

to be resistant

(1)

Beauchamp

of sodium

is a

33,000

bovine

molecular

that one of the isozymes

liver

from

it has been found (2).

1.15.1.1)

The copper

of 2-mercaptoethanol

in the presence

Since

of zinc.

The enzyme

are present

observed

liver

into monomers

(E.C.

and it has been found

of the protein,

disulphide

swordfish

of approximately

and 2 atoms

studied

Monomers

dismutase

weight

in the enzyme.

dissociation.

we have

superoxide

of copper

has been extensively

the sequence

from

active.

: Copper-zinc

dimeric

of Malta

-Malta

Summ ax-y:

which

H . Bannister

of

that the bovine

of 10 M urea

on the enzyme

from

(51,

we

swordfish

liver.

,M aterials liver

and Methods:

of the swordfish

described

(4).

Copper-zinc (Xiphias

Solutions

superoxide

gladius)

dismutase

was prepared

of the enzyme

469

(30 mg’/ml)

from

the

as previously were

dialyzed

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 8 1, No. 2, 1978

BIOCHEMICAL

40

50 Fraction

Fig.

1.

A,

indicate

and (c)

Fig.

2.

(A-A)

Fridovich

volume

(b)

(O-O)

protein

15

= 1.8 ml.

chymotrypsinogen

native

concentration

(6)

pH 7.4 containing

Am berlite

. Chromatography AC 54 (LKB buffer.

augmentation

photooxidation monitored

ovalbumin,

Time 10 (Min)

in 8 M urea

Fraction

reaction.

Final

buffer

with

the dialysis

photochemical

oxide

of the assay

enzyme.

Ultrogel with

dismutase

pH 7.4.

of (a)

5

enzyme in the cuvette

mg/ml.

England)

brated

Results

course

was de-ionized

containing

0

c.

0.1 M phosphate

Poole,

buffer,

peaks

cytochrome

Time

was 0.027

urea

elution

monomeric

against

of 30 mg superoxide

0.1 M phosphate

Arrows

02

60

No.

Gel filtration

containing

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

which

takes

of dianisidine

M onobed

dismutase

behaves

AB,

Enzyme

assay advantage

as a dimer

Bromma,

described

by Misra

by riboflavin.

to be unaffected

state

470

equiliby a and

of aerobic

The reaction

is

by 8 M urea.

swordfish

of 32,500

90 x 1.5 cm

Sweden)

rate

The

( B .D . H . Ltd. ,

was measured

of the increased

sensitized

In the native

Resin

at 4OC.

out on a column

activity

recently

at 460 nm and was found

and Discussion:

MB-3

was carried Produkter

8 M urea

molecular

copper-zinc weight

superwith

the

20

BIOCHEMICAL AND BIOF’HYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2, 1978

0 0

12

6

16

Protein

Fig.

3.

native

Reaction

rate

enzyme,

activity

@-A)

in 8 M urea

same

activity

native

cular

and 50% glycerol

enzyme

dialyzed

against

into monomers

weight

of 16,300. the activity

monomer

tended

to show

concentration

lower reaction

lower

These

might

show

cycling

Fielden

--et al. (8) A detailed kinetic superoxide

AC 54 of

that the enzyme patter

a mole-

(in 8 M urea)

was

and the

of both time concentration

than the dimer of the medium

readily

indicated

At low protein

indicate

be able to act independently of superoxide

redox

on Ultrogel

as a function

as similarly

investigations

the dismutation that

activity

lowered

(Fig. ti

and the

3) . This

the fact that the

low activity

was present

in

3).

preliminary

the enzyme

concentration.

. Both the dimer

activity

is due to the viscosity

(Fig.

protein

of the monomer

2 and 3).

(7)

indicate

indicates

dimer

(O-O)

Arrows

The elution

the same

is diffusion-limited

50% glycerol

8 M urea

30

concentration.

Gel filtration

of native

(Figs.

had a slightly activity

(4).

The activity

with

monomer

at low

(Fig.1).

compared

protein

enzyme.

24

(pg/ml)

of protein

monomeric

as bovine

enzyme

dissociates

as a function

concn.

radicals.

of the copper

is not an absolute study

dismutase

of monomeric

that the two active of each other

The results in dim eric necessity

471

enzyme

when also

swordfish

in

catalyzing seem

, as proposed

for the dismutase

and dimeric

is in preparation.

would

sites

activity. liver

to by

Vol. 8 1, No. 2, 1978

BIOCHEMICAL

Acknowledgements and helpful financial

: We thank

comments

:

1.

B.B.,

Keele, Chem., Abernethy,

5.

Irwin

thanks

Fridovich

the Wellcome

for advice Trust

for

Steinman,

J.L.,

C .O.

I.,

(1971)

H.M.

R.L.

J. Bid.

andHill,

(1974)

J. Biol.

and Fridovich,

I.,

(1973)

Biochem

and Bannister,

W.H.

. Biophys.

50-56.

Bannister,

J.V.,

Biochem

. Physiol.,

Forman,

and Fridovich,

7339-7347.

Beauchamp, 317,

J.M.

2875-2880.

249,

Acta, 4.

W.H.B.

McCord,

246,

Chem., 3.

Professor

support.

References

2.

.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H.J.

Anastasi, 56B,

A.

(1977)

Comp.

235-238.

and Fridovich,

I.,

(1973)

.I. BioI.

Chem.,

248,

2645-2652. 6.

Misra, 181,

H.P.

and Fridovich,

I.,

(1977)

Arch.

Biochem.

Biophys.,

308-312.

7.

Fridovich,

I. , (1974)

8.

Fielden,

E.M . , Roberts,

Mautner, J ., 139,

G.N.,

Rotilio,

Adv.

Enzymol.,

P.B.,

Bray,

41, 35-97. R.C.,

G. and Calabrese,

49-60.

472

Loewe, L.,

D.J.,

(1974)

Biochem.

Active subunits from superoxide dismutase.

Vol. 81, No. 2, 1978 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 469-472 March 30,1978 ACTIVE SUBUNITS Joe V. Bannister, Dept. F...
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