Evidence for superoxide generation by NADPH-cytochrome C reductase of rat liver microsomes

BIOCHEMICAL

Vol. 47, No. 5, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EVIDENCE FOR SUPEROXIDE GENERATION BY NADPH-CYTOCHROME C REDUCTASE OF RAT LIVER MICROSOMES] S.D.

Aust2,

D.L.

Roerig

and T.C.

Pederson

Department of Biochemistry Michigan State University East Lansing, Michigan 48823

Received

April

28, 1972

Rat liver microsomes are capable of catalyzing an NADPH-dependent oxidation of epinephrine to adrenochrome that is inhibited by superoxide dismutase. Activity is greater and more sensitive to inhibition by superoxide dismutase at pH 8.5 than pH 7.7. The epinephrine oxidation activity copurifies with NADPH-cytochrome c reductase. Two analytical ration

of the

of epinephrine is

tools

superoxide

of the

by McCord

and Fridovich

dismutase

to study

numerous

flavoproteins,

fact

that

absence

of substrate

oxide

(4,

superoxide

generation

(3)

(1).

partially

oxidation The second

of erythrocuprein superoxide

of cytochrome

c by

microsomes

comes from the

oxygen

in the

and Coon (6)

and ethylmorphine

and a solubilized,

anion

have used

an NADPH-dependent

5) and Strobe1

on the

reduction

by liver

the gene-

based

activity

et al.

dependent

catalyze

to investigate

an assay

dismutase

Massey

the oxygen

hydroxylation

generator

by the

(2).

possible

One is

superoxide

in superoxide

microsomes

benzphetamine

anion.

to adrenochrome

the discovery

Interest

have made it

uptake

have demonstrated

demethylation purified

using

preparation

a superof

1 This study was supported in part by EPA Grant No. 8 ROl EP00801 and NIH Training Grant GM 1091 from the National Institute of General Medical Sciences. 2 To whom inquiries

should

3 Present address: Wood, Wisconsin

Clinical 53193.

be addressed, Pharmacology

1133 Copyright@1972,by Academic Press,Inc.

Services,

Wood V.A.

Hospital,

Vol. 47, No. 5, 1972

cytochrome oxide

BIOCHEMICAL

Pd50

(7,

generation

activity

8).

This

by rat

copurifies

communication

liver

with

rats

described

and washed

once in 0.1

NADPH-cytochrome

(10). Company, under

San Jose,

Calif.;

N2) and purified The freshly

( 11). c/min/mg 2.10

* lo*

c&/mmole

Epinephrine

of Mazur

--et al.

(1).

xanthine

oxidase

(Grade

I from

and epinephrine

dismutase

nificant by rat

Neither

anaerobiosis

liver

microsomes.

microsomes

do catalyze

adrenochrome

(Fig. and is

microsomes

an unexplained

rig/ml

reduction

lag.

dismutase superoxide

of 25 rig/ml

microsomes

(28 kg/ml,

on epinephrine

tetrazolium

reduction

The rate could

was highest dismutase

c by xanthine superoxide pH 8.5)

was higher

higher

oxidase

was inhibited

oxidation. 1134

also

to

concentration

of

the

pH (Fig.

Boiled

by

than

at

sensitivity

1).

oxygen

oxidation

9%.

characterized

Also

and xanthine.

the

had

However,

at pH 8.5

55% of the

dismutase

c catalyzed

of epinephrine

were

at pH 6.5.

inhibited

had a sig-

dismutase

on the

The assays

at the

tetrasuperoxide

by microsomes.

dependent

be observed

Co.;

of cytochrome

oxidation

of oxidation

NADPH,

blue

dismutase

and superoxide

by boiling.

by the

Type IV), nitro

superoxide

an NADPH-dependent is

heart,

c as

Inc.

reduction

Activity

cytochrome

was assayed

Laboratories,

Anaerobiosis

destroyed

of cytochrome

presence

effect

1).

No oxidation

to superoxide 2.5

blue

for

xanthine,

nor

at 4'

of cytochrome

from Sigma Chemical

on the NADPH-dependent

on nitro

PH 7.7.

obtained

from Miles

effect

no effect

were

was obtained

RESULTS:

31 pmoles

c (beef

buttermilk),

16 hrs

(Dole

of Omura and Takesue

oxidation

Cytochrome

M sucrose

Bromelain

protein,

by the methods

of

as previously

with

reduce

the livers

and 0.3

on e55o (reduced-oxidized)

method

zolium,

phenobarbital

M pyrophosphate

enzyme would

(12).

super-

generation

from

was solubilized

and assayed

based

isolated

10 pg/mg microsomal

purified

protein,

were with

c reductase

for

c reductase.

Microsomes pretreated

evidence

The superoxide

NADPH-cytochrome

and from rats

(9)

presents

microsomes.

METHODS AND MATERIALS: control

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

At pH 8.5,

dependent In the

of epinephrine dismutase

by had no

BIOCHEMICAL

Vol. 47, No. 5, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pH 7.1

.08

z 2.04 t 8

10

5

TIME

(MINI

Fig. 1. Oxidation of epinephrine to adrenochrome by rat liver microsomes. Reaction mixtures contained 28 bg/ml microsomal protein, 5 x 10m4 M epinephrine, 1 x 10m4 M EDTA and 1 x lo- 4 M NADPH buffered at pH 7.7 or 8.5 in 0.15 M potassium phosphate. Microsomes were isolated from the livers of phenobarbital treated male rats. Optical density was recorded in a Coleman model 124 spectrophotometer at 480 v. Superoxide dismutase (SDM) was added at 5 kg/ml.

NADPH-cytochrome purified

by the method

ability were

c reductase

to generate conducted

c reductase epinephrine)

of Omura and Takesue

superoxide

at the

and reduce

same pH and ionic

to superoxide remained

was solubilized

generation

constant

(11)

Bromelain

and assayed

cytochrome strength,

(as judged

throughout

with

c.

for

When all

the ratio by ability

purification

and its assays

of cytochrome to oxidize

(Table

1).

In

Table 1. NADPH-dependent reduction of cytochrome c and oxidation of epinephrine by microsomes and NADPH-cytochrome c reductase isolated from the livers of phenobarbital treated rats. The NADPH-dependent reduction of cytochrome c (11) and the oxidation of epinephrine (1) were assayed in a Coleman Model 124 spectrophotometer at the indicated wavelength in 0.15 M phosphate buffer, pH 8.5, 22'C. Enzyme

A. Cytochrome c reduction ~moles/min/mg

Microsomes Enzyme from DEAE column

first

Enzyme from DEAE column

second

B. Epinephrine oxidation ~moles/min/mg

A/B

0.2

0.07

2.85

7.1

2.5

2.84

16.0

4.75

3 .16

1135

Vol. 47, No. 5,1972

addition tion

BIOCHEMICAL

the

ratio

of NADPH-cytochrome

in microsomes

to that

isolated

obtained

pretreated

with

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

the

microsomes

phenobarbital

c reduction livers

from

to epinephrine

of control

the liver

(Table

rats

of rats

oxida-

was identical

that

had been

2).

Table 2. NADPH-dependent reduction of cytochrome c and oxidation of epinephrine by microsomes isolated from the livers of control and phenobarbital treated rats. The NADPH-dependent reduction of cytochrome c (11) and the oxidation of epinephrine (1) were assayed in a Coleman Model 124 spectrophotometer at the wavelengths indicated in 0.15 M phosphate buffer, pH 8.5, 22'C. Microsomes

B. Epinephrine oxidation ~moles/min/mg

A. Cytochrome c reduction ~moles/min/mg

A/B

Control

0.11

0.04

2.75

Induced

0.21

0.075

2.80

DISCUSSION: ration

of superoxide

microsomes tion

Evidence

is

as expected occurs

by rat

are capable

activity

since

reductase.

Also,

oxidation

the

pretreated

suggest

that

the

with

is

activity

ratio

at

copurifies

from

phenobarbital

superoxide

higher

the

remains

generation

the

high

pH.

at high

pH,

anion

sensitivity

to

NADPH-cytochrome

c reduction

livers

Oxida-

superoxide

Also

with

of NADPH-cytochrome

isolated

more active of the

(13).

lowered

dismutase

in microsomes

animals

to pH, being

gene-

of NADPH

to adrenochrome.

dismutation

as the pH is

an NADPH-dependent

In the presence

epinephrine

the non-enzymatic

oxidation

for

microsomes.

sensitive

by superoxide

Epinephrine

liver

of oxidizing

extremely

more rapidly

inhibition

has been presented

to epinephrine

of control constant.

animals These

can be ascribed

c

and

results

to NADPH-cytochrome

c reductase. There

has been some question

in the microsomal their laurate

hydroxylation

detergent-solubilized hydroxylation

as to the system.

Lu,

NADPH-cytochrome when combined

with

1136

their

identity Junk,

of the

flavoprotein

and Coon (14)

c reductase cytochrome

would P-450

found support fraction

that

BIOCHEMICAL

Vol. 47, No. 5, 1972

in the presence

of lipid,

but

of Williams

and Kamin would

a reductase

fraction

cytochrome

c would

trypsin.

Several

NADPH-cytochrome

that only

c reductase

in this

pholipids

substitute

will

study for

inhibit

will

--et al.

c after

brief antisera

hydroxylations Dr.

(15)

reduce

the detergent-solubilized catalyze

found

that

and reduce

incubation

with

against

purified

by intact

M. J. Coon we have

anaerobically

also

reductase

hydroxylation

shown that

with

The enzyme will

micro-

found

cytochrome reductase

the peroxidation

that P-450,

in drug of phos-

(19).

ACKNOWLEDGMENT: for

have

Ichihara

laurate

cytochrome

investigators

the enzyme used

hydroxylation.

support

reduce

lipase-solubilized

the pure, However,

would

In collaboration

not

that

not.

somes (15-17).

but will

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

collaborative

We are grateful

experiments

to Dr.

involving

M. J.

cytochrome

Coon and H. W. Strobe1 P,,o

reductase

assays.

REFERENCES: 1. ;: 4. 2: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Mazur, A., Green, S. and Shorr, E., J. Biol. Chem., 220, 227 (1956). McCord, J.M. and Fridovich, I., J. Biol. Chem., 2& 6049 (1969). Massey, V., Strickland, S., Mayhew, S.G., Howell, L.G., Engel, P.C., Matthews, R.G., Schuman, M., and Sullivan, P.A., Biochem. Biophys. Res. Commun., & 891 (1969). Orrenius, S., Ericsson, J.L.E. and Ernster, L., J. Cell Biol., 2, 627 (1965). Orrenius, S,, J. Cell Biol., 5 713 (1965). Strobel, H.W. and Coon, M.J., J. Biol. Chem., 246, 7826 (1971). Lu, A.Y.H., Strobel, H.W. and Coon, M-J., Biochem. Biophys. Res. Comn., s 545 (1969). Lu, A.Y.H., Strobel, H.W., and Coon, M.J., Mol. Pharmacol., 6, 213 (1970). Pederson, T.C. and Aust, S.D., Biochem. Pharmacol., 19, 2221 (190). Sachs, H., Biochim. Biophys. Acta, 2l, 188 (1956). Omura, T. and Takesue, S., J. Biochem., 67, 249 (1970). Massey, V., Biochim. Biophys. Acta, 3& 255 (1959). Knowles, P.F., Gibson, J.F., Pick, F.M. and Bray, R.C., Biochem. J., 111, 53 (1969). Lu, A.Y.H., Junk, K.W. and Coon, M.J., J. Biol. Chem., 244, 3714 (1969). Ichihara, K., Kusunose, E. and Kusunose, M., FEBS Letters, a 105 (1972) * Wada, F., Shibata, Biophys. H., Goto, M. and Sakamoto, Y., Biochim. Acta, 162, 518 (1968). Glazer, R.I., Schenkman, J.B. and Sartorelli, A.C., Mol. Pharmac., ‘7, 683 (1971). Masters, B.S.S., Baron, J., Taylor, W.E., Isaacson, E.L. and LoSpalluto, J., J. Biol. Chem., 246, 4143 (191). Pederson, T.C. and Aust, S.D., Fed. Proc., & 894 (1972).

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