Separation of aldehyde reductases and alcohol dehydrogenase from brain by affinity chromatography: Metabolism of succinic semialdehyde and ethanol

BIOCHEMICAL

Vol. 63, No. 4, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SEPARATION OF ALDEHYDE REDUCTASES AND ALCOHOL DEHYDROGENASE FROM BRAIN BY AFFINITY CHROMATOGRAPHY: METABOLISM OF SUCCINIC SEMIALDEHYDE AND ETHANOL Boris

Tabakoffn and Medizinisch-chemisches University of Berne,

Received

February

25,

Jean

P.

von Wartburg lnstitut Switzerland

Berne,

1975

Enzyme activities which reduced aldehydes such as succinic semialdehyde in rat brain cytosol were separated by affinity chromatography on NADP-Sepha rose. The reduction of succinic semialdehyde which was previously thought to be catalyzed by alcohol dehydrogenase or lactate dehydrogenase was shown to be primarily catalyzed by enzymes both separable from and characteristically different from these enzyme activities. Brain alcohol dehydrogenase activity was shown to be
tissue

of

aldehyde

dehydrogenase which

of

aldehydes used

brain

to

characterize

alcohol

dehydrogenase

to

the

may

(10)

attributed

action

of

(7,l).

various

pyridine

attribute

the It

was

Address

:

succinic

a preparative

procedure dependent

also

of of

particular

for

been of

957

as

to

have

also

sub-

substrate

partially

of purified

(8)

succinic

However,

of

which are

reported

that

brain

semialdehyde a previous

semialdehyde

in

brain

report to

the

1.1.1.28). by

which

we

could from

aldehydes

particular

posed

a

oxido-reductases

The Chicago Medical Chicago, Illinois

o 19 75 by Academic Press, Inc. of reproduction in any form reserved.

(3)

substrate

alcohol

metabolism

acetaldehyde

has

as

lactaidehyde,

(9).

(E.C.

well

reductive

conversion

dehydrogenase

metabolism

Present

the

of

nucleotide

proteins.

it

molecular

been

reductase

as

Recently

reduction

as

and

y-hydroxybutyrate

the

the

such

serve

catalyzes

hypnotic

lactate

for

(6)

as have

aldehyde

also

multiple

(1,2,3)

responsible Aldehydes,

dehydrogenase

contain

questions

dehydrogenase

reductase

known

and

brain

alcohol

We attempted

Copyright AN rights

is

(5).

in

to

(E.C.1.1.1.2) (4)

enzymes

for

alcohol

demonstrated

reductase

strates

aldehyde

been

(E.C.l.l.l.1)

class

been

has

to

interest

to

School, 60612

2020

the

obtain W.

separate brain

separated a preparation Ogden

Avenue,

and enzyme of

Vol. 63, No. 4,1975

BIOCHEMICAL

alcohol

dehydrogenase

ethanol

by

direct

dehydrogenase duction ethanol

has

been

in

lactaldehyde to

wherein

acetaldehyde

in

to assay

one

could

analysis.

brain

to

demonstrated

lactaldehyde

brain

spectrophotometric activity

of

of

from

AND BIOPHYSICAL

have

enzyme

by

I iver

be

horse increased

mixtures

measure Previous

utilized

estimate

RESEARCH COMMUNICATIONS

an

nearly

of

coupled

to

(4,ll).

alcohol

oxidation

studies

assay

activity

the

The

fold

by

alcohol the

re-

conversion

dehydrogenase,

100

of

however,

the

inclusion

of

(12).

MATERIALS AND METHODS Adult male rats of the CFR strain were decapitated, brains were removed, washed, and a 20% homogenate was prepared in 10 mM sodium phosphate pH 8.0. The homogenate was centrifuged at 106,OOOxg for 50 min and the clear supernatent fluid was recovered. This fluid was fractionated using ammonium sulfate. Protein precipitating between 35 and 70% saturation with ammonium sulfate was recovered and desalted by gel filtration chromatography on Sephadex G-25. Column eluate which contained protein was pooled and concentrated using an Amicon filter apparatus. This concentrated solution will be referred to as the ammonium sulfate fraction. The ammonium sulfate fraction was further fractionated using affinity chromatography on NADP linked Sepharose. Fractions eluted from this column which metabolized aldehydes (see legend Fig. 1) were pooled, concentrated, and further characterized. “Coupled” assays of alcohol dehydrogenase activity (12) were performed as described by Raskin $nd Sokoloff (4). Reaction mixtures-contained Llactaldehyde (8mM) , NAD (0.5 mM) , ethanol (200 r&l) and enzyme in 50 ti sodium phosphate pH 7.4. Mixtures were incubated at 37’ for 30 min and the propanediol produced from lactaldehyde was quantified as described by Jones and Riddick (13). “Direct” assays of alcohol dehydrogenase activi ty+were performed by measuring spectrophotom$trically the conversion of NAD to NADH in reaction mixtures containing NAD (5 mM), ethanol (16 mM) and enzyme in 100 mM sodium pyrophosphate pH 8.8. Reduction of aldehydes was monitored spectrophotometrically in mixtures enzyme, and aldehyde substrate in 100 mM containing NADH or NADPH (0.16 mM), All incubation mixtures were one ml in total volume sodium phosphate pH 7.4. and spectrophotometric assays were performed at 25’. L-lactaldehyde was prepared from D-threonine as described by Huff and Prior to use the pH of the solutions was adjusted to pH 7.4 Rudney (14) . and lactaldehyde was dedimerized by heating at 80’ for 10 min. Succinic semialdehyde was prepared from hydroxybutyrolactone as deThe presence of succinic semialdehyde was scribed by Taberner et al (15). determined as described by Salvador and Albers (16). The quantity of this and other aldehyde substrates in solutions was assertained using horse liver a I cohol dehydrogenase (1) . NADP-Sepharose was synthesized by the procedure of Larson and Mossbach Protein was determined by the method of Lowry et al (18) with bovine (17) . serum albumin as the standard. RESULTS

Rat

in

Table

inhibitor and

brain

cytosol

1 in

the

presence

of

alcohol

acetaldehyde

catalyzed

the

of

NADPH

either

dehydrogenase reduction

in

(lg), the

reduction or had

presence

958

of NADH.

little of

NADH

the

aldehydes

Pyrazole, effect could

a known on

not

listed

this be

activity detected.

Vol. 63, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL

TABLE REDUCTION

OF ALDEHYDES

I BY

RAT

SUBSTRATE p-nitrobenza

(5

L-lactaldehyde (2 x

CYTOSOL

NADPH

NADH

12

NADPH

59

NADH

55

NADPH

I1

lo-3rj)

Succinic

(5

BRAIN

COFACTOR

dehyde 10’ 1 M)

x

RESEARCH COMMUNICATIONS

Semialdehyde 10-4M)

x

5

NADH

Enzymatic in the

Acetaldehyde (1 x

lo-3M)

activity

was

monitored

spectrophotometrically

as

Incubation produced

3.

n.d.

of reaction 15% inhibition

= not

Alcohol

dehydrogenase

strated

by

of

lactaldehyde.

the

presence

reported

the The

of +

under

activity of

rate

4.4

by this Fractionation

mU/gm

Raskin

of

alcohol

found by

cytosol the

to

be

brain

cytosol

These

values

Sokoloff dehydrogenase

with

brain

ammonium

(4)

and

with

pyrazole

(1mM)

however,

be

demon-

ethanol

to

reduction

oxidation

of

reduction

which

was

13.8

mU/gm

brain

NAD+

in

+ and

are

similar

ourselves

the to

(20).

dependent

performed

absence those

The of

as

ethanol

previously

Pyrazole

so

on

(n=5).

(I

98%. was

959

of

could,

25.8

activity sulfate

activity obby the and NADPH multip-nitrobenzalde-

divided

conditions.

lactaldehyde

brain. and

assay

coupling

was

lactaldehyde

our

in

assay

ethanol

specific

mixtures in the presence of enzyme activity.

detectable

means

of

reduction

13.6

described

text.

2.

was

3

n.d.

Relative specific activity was taken as the tained with the various aldehydes and cofactors specific activity obtained with p-nitrobenzaldehyde plied by 100. The specific activity obtained hyde and NADPH was 10.3 mU/mg protein.

1.

hibited

NADH

to

maximize

mM)

in-

Vol. 63, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL

I

Pod

II ’

’

Pod

RESEARCH COMMUNICATIONS

III

Pool

IV

’

!.I

1.0

i . 15 5 P ; l.Of E 0

Q5

0

the

recovery

Approximately

of

alcohol 40%

of

dehydrogenase the

activity

activity was

recovered

960

measured with

by

the

a purification

coupled

assay. of

two

BIOCHEMICAL

Vol. 63, No. 4,1975

fold

by

this

with

this

procedure. fraction

However,

in

the

ing

aldehydes

(Fig.

1)

coupled of

alcohol

The

of

the

ammonium

sulfate

several

peaks

protein the

the

ranged to

activity

protein.

to

enzyme

Pool II

II.

assay using

mU/mg

the

specific

protein

to

and of

56 +

brain

to

of

rat

reduction

of

lacfaldehyde

6. oxidize was

II

25.8

mU/mg

less

than

assay

for

0.37

mU/mg

on ethanol

approximately

the

of

alcohol

in dehy-

protein

direct

these

12%.

2 mU/mg

using

activities Based

by

pooled

a recovery was

the

reduc-

purification

activity

using

specific

be

capacity

of

of were

overall

to

direct

re-

capable

with

compared the

NADP-Sepharose

measured

Pool

activity

ratios

found

in as

fraction

2.4

fold

aldehydes

aldehydes

The

18

was

of

activity

activity

a specific

The were

the

II,

on

dehydrogenase

hand,

sulfate to

of

dehydrogenase

other

reduction

reducing

Pool

coupled

Pool

0.54

mU/mg

that

the

ammonium

preparations

calculated

alcohol the

in

from

0.04

identical

enzyme

of

the

fraction

in

in

RESEARCH COMMUNICATIONS

fractions

localized found

On

activity In

various

alcohol

by

pools.

drogenase

0.01

uhen

primarily

measured

other

assay

I).

activity as

found.

of

dehydrogenase

specific

pyrazole.

by

concentrated, was

cytosol,

inhibited

separation

assay

in

not

(Fig. and

as

was

Chromatography sulted

AND BIOPHYSICAL

was the

coupled

assay

from

measured

in

figures, without 0.45

we coupling mU/gm

brain.

Figure

1 CHROMATOGRAPHY

OF ALDEHYDE REDUCING ON NADP-SEPHAROSE

ENZYMES

Columns were developed using 100 ml of 5 mM sodium phosphate pH 8.3 followed by a linear gradient of potassium chloride in the same buffer. Conductivity of collected fractions (s.*.) is expressed as IQ-T.cm-I. Fractions (2.5 ml each) were assayed for enzyme activity as described in the text. Upper frame: (A-A-A )enzyme activity with L-lactaldehyde and NADH as cosubstrates; b-e-0) activity with L-lactaldehyde and NADPH as cosubstrates; @CU)activity with p-nitrobenzaldehyde and NADPH as cosubstrates. Lower frame: (A-A-A ) activity with succinic semialdehyde activity with succinic semialdehyde and NADPH as cosubstrates; (0-O-O) and NADH as cosubstrates; @r-8) activity with p-nitrobenzaldehyde and NADPH as cosubstrates. Protein in both frames denoted by (- - - ). Fractions were pooled as denoted below the abscissa, concentrated when necessary and used for inhibitor studies summarized in Table 2.

961

assay)

assay)

semialdehyde

NAD+

NAD+

NADPH

15

n.d.

-

-

31

c

0

0

14

Fractions elluted from NADP-Seph# ‘ose were pooled as or NADPH as cofactors was assayed as described under “direct” assays was also assayed as described in the in each pool. not detectable under our assay conditions. I. n.d.=

ethanol (coupled

ethanol (direct

succinic

64

35

7

NADPH

dehyde

NADPH

i t robenzal

L-Tactaldehyde

p-n

-

14

38

NADH

succinic

n.d.

0

I7

10

NADH

p-nitrobenzaldehyde

semialdehyde

22

3

479

Pyrazole

NADH

I

shown methods. methods

in

3

5

5

23

0.4

n.d.

n.d.

24

ACTlV

ACTIVITIES

figure Ethanol section.

0

20

0

0

63

Oxalate

ON ENZYME

% INHIBITION

POOL

INHIBITORS

Pheno

OF

L-lactaldehyde

EFFECT

ACTIV

2

COFACTOR

SUBSTRATE

Table

94

99

D

-

8

0

IO

57

Oxalate

I

0.8

-

-

85 35

n.d.

86

4

510 35

-

-

Pheno

274

28

n.d.

n.d!

ACTIV

NADP-SEPHAROSE

III

4 4

2

21

-

Pyrazole

% INHIBITION

POOL

NADH 1 and reduction of aldehydea with either oxidation using either the “coupled” or Activity is expressed as total mU recovered

12

0

90

1

79

Pyrazole

26

0

Pheno

I I

FROM

% INHIBITION

POOL

ELUTED

-

Oxalate

0

0

0

3

Vol.

63,

No.

4, 1975

That

this

alcohol

importance

in

Fig.

Table

1 and

the

semialdehyde enzyme

BIOCHEMICAL

not

inhibited

reducing

activity

was

(21). brain

inhibitor

of

of

and

semialdehyde

in

activity

is

and

and

the

of

in

IV.

Pool

(>gO% zole

different

previously

portion

was

This

presence

separable

from

aldehyde

activity

was I

quite

mM sodium

oxalate,

and

was

nearly

pyruvate

re-

to

an

of

both

alcohol

(1,2,27).

The was

by

was

this

dehydrogenase

inhibition

exclusively

These

that

activity

but

reduc-

metabolizing

indicate

reductase

phenobarbital)

lactate

phenobarbital.

activity

sensitive

of

activity

of

I was

However,

NADPH

bulk

aldehyde

Pool

pools

inhibit

by

enzyme

reductase

these

80-85%.

and

the

in

reduction

affected

NADH

of

Phenobarbital,

III

of

of

to

the

less

profile

described

with

sodium

much

portion

in

studies.

Pool

by succinic

inhibitor

found

primary

recovered

found

inhibited

elution

the

previously

inhibition or

present

with

described

the

the

major

was

was

of

sensitive the

a known

&Ij

(22),

the

II

not

illustrated

pyrazole

activity

(5

in

semialdehyde

succinic

Pool

oxalate,

oxalate *95%

and

in

COMMUNICATIONS

is is

semialdehyde

sodium

reductase

patterns

of

noted

p-nitrobenzaldehyde

succinic

inhibition

Sodium cytosol

amount

reductase or

aldehyde

lactaldehyde tion

pyrazole

brain

semialdehyde

succinic

semialdehyde

RESEARCH

from

succinic a small

by

by

of

metabolizing

BIOPHYSICAL

activity

Although

dehydrogenase duction

metabolism

Succinic

111.

dehydrogenase

2.

activity

and

AND

recovered

phenobarbital

unaffected

dependent

major

by

on

pyra-

NADPH

as

a

led

to

cofactor. DISCUSSION

The

speculation in

that

redox

reports

have

these

hand,

Veloso

redox

couples

of

(i.e. on

changes changes et

al after

However

of

ethanol

alcohol

dehydrogenase

in

tissue

this

lactate/pyruvate)

appeared

redox

buted

presence

oxidation

couples

nificant

anesthesia.

possible

this in

to

brain

after

the

activity

the Rawat

administration

of

to

in

(23,24).

attributed

ethanol ,

matter

oxidation

(24)

as

ethanol the

small

changes

in of

by

alcohol

963

brain

changes pC02

in

would

produce

liver. al

of

ethanol

(23)

found and

tissue.

On

they

found

by

ethanol

dehydrogenase

changes

Contradictory

et

caused

brain

was

the in

sigattriother brain

induced not

measured

Vol. 63, No. 4,1975

directly

in

BIOCHEMICAL

these

dehydrogenase

studies.

provided

genases

and

alcohol

dehydrogenase

support

the

that activity

in

our

activity

photometric

the

oxidation

in

the

production

Since

rat

horse

enzyme

would

be

by

of

The

responsible

butyrate

(8)

dehydrogenase

the

of had

dehydrogenase a cofactor from

(Table previously

II

1)

Greater

and

the

little

contamination

aldehyde

reductase

form

localized

primarily

in

succinic by

rate

of

the alcohol spectro-

of

lactaldehyde fold

no quite

increase

lactaldehyde(l2). similar

limiting

to

(NADH

(I)

activity to

studies.

Al though

succinic

catalyzing

the

off)

the

step

different

enzymatic

activity

was

since

this

has

Deitrich,

preparation).

964

found

alcohol the

succinic alcohol

with

reductases would

mitochondria.(Ris,

the

NADPH

differed

preparation enzyme

of from

inhibitors and

major

y-hydroxy-

reduction

quite

to

the

semialdehyde,

properties

our

is

semialdehyde our

dehydrogenases of

4.2

the

intermed-

alcohol

a 100

be

in

liver

reduction

to

low

of

“direct”

horse

dehydrogenase

sensitivities

described

addition,

that

metabolized

activity

1).

by

containing

shown

supported

chromatographic

(Fig.

been

of

Pool

enzymatic

the

use

value

approximately

alcohol

not

in

change

greater

of

assays

This

The

obtained

predicted

assay.

brain

was

activity

semialdehyde

coupled

conversion

brain

portions

in

expect

any

of

would

who

1 mU/gm.

to

fold

coupling

we

for

a 50

over

should

little

brain)

(24)

<

dehydro-

estimate

rat

Veech

alcohol

other

a direct

administration.

resulted

that

in

assay

has

in

add

make

was

brain

from

mU/gm

measurements

dehydrogenase

contention

enzyme

the

brain

estimates

acetaldehyde

(25,26)

bypassed

to

ethanol of

alcohol

in

Previous

activity

to

and

produced

compared

measurements.

dehydrogenase

Passonneau

ethanol

studies

to

(0.45

would

after

us

rat

enzyme

results

ethanol

brain

of

this

allowed

Our

dehydrogenase in

dehydrogenase

separating

Veloso,

RESEARCH COMMUNICATIONS

purification

and

oxidizing

alcohol

assay

of

brain

of

activity

“coupled”

partial

activity.

metabolites

ably

in

contention

of

major

a means

reductases

enzyme

iary

Our

AND BIOPHYSICAL

been von

consider(Table

be

2).

expected shown

Wartburg:

as

from to

be in

In

Vol. 63, No. 4,1975

Fishbein

and

responsible

for

However

, sodium

effect

on

we

the

can

enzymes

be would

further

succinic

be

portion

y-hydroxybutyrate means

of

for

lactate

of

affinity

in

the

had

Sodium of

undescribed

chromatography.

Since

properties

known

is

in

did,

NADH.

reducing

previously

a

little

oxalate

presence activity

of

was

y-hydroxybutyrate.

2).

enzymatic

production

their

dehydrogenase

dehydrogenase

(Table

to

the of

lactate to

lactaldehyde

to

responsible

characterization

of

major

by

of

semialdehyde

the

separated

brain

RESEARCH COMMUNICATIONS

semialdehyde

inhibitor

metabolism

ascribe

that

succinic

a known of

AND BIOPHYSICAL

proposed

of

oxalate

semialdehyde

which

(10)

conversion

inhibit Thus,

brain

Bessman

metabolism

however,

succinic

BIOCHEMICAL

enzymes these

sommifacent progress

in in

our

laboratories. Acknowledgements: assistance. Alcohol Abuse Stroke, Swiss and Deutsche

We would 1 ike to thank Maria Kaufmann for Supported in part by grants from USPHS National and Alcoholism, National Institute of Neurological National Science Foundation and Hoffman-LaRoche Forschungsgemeinschaft. BT is Schweppe Foundation

her fine technical institute of Disease and Research Fdn. Fellow.

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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21.

Ris, M.M., von Wartburg, J.P. (1973) Eur. J. Biochem. 37: 69-77. Tabakoff, B., and Erwin, V.G. (1970) J. Biol. Chem. 245 3262-3268. Turner, A.J. and Tipton, K.F. (1972) Biochem. J. 130765-772. Raskin, N.H. and Sokoloff, L. (1970) J. NeurochemT7: 1677-1687. Pietruszko, R. (1974) Alcohol and Aldehyde Metabolizing Systems, Eds. Thurman, R.G. et al, 529, Academic Press, New York. see also von Wartburg, J.P., (discussion) pg. 549-552, same volume. Huff, E. (1961) Biochim. Biophys. Acta 48: 506-517 Tabakoff, B., Vugrincic, C., Anderson, R.A. and Alivisatos, S.G.A. ,(1974) Biochem. Pharmacol. 23: 455-460. Taberner, P.V. (1974miochem Pharmacol. 23: 1219-1220. Giarmann, N.J. and Roth, R.H. (1964) Science 145: 583-584. Fishbein, W.N. and Bessman, S.P. (1964) J. Bix Chem. 239: 357-361. Raskin, N.H. and Sokoloff, L. (1968) Science 162: 131-132. Gupta, N.K. and Robinson, W.G. (1966) Biochimnophys. Acta 118: 431-434. Jones, L.R. and Riddick, J.A. (1957) Anal. Chem. 29: 1214-1217. Huff, E. and Rudney, H. (1959) J. Biol. Chem. 234~1060-1064. Taberner, P.V., Barnett, J.E.G. and Kerkut, G.r(l972) J. Neurochem. -19: 95-99. Salvador, R.A. and Albers, R.W. (1959) J. Biol. Chem. 234: 922-925. Larsson, R-O. and Mosbach, K. (1971) Biotech. Bioengin.3: 393-398. Lowry, O.H., Rosebrough, N.J., Farr, A.J. and Randall, Rx (1951) J. Biol. Chem. 193: 265-275. Theore??, H. andonetani, T. (1963) Biochem. Z. 338: 537-553. von Wartburg, J.P., Berger, D., Ris, M.M. and Tabakoff, B. (1975) Experimenta Med. and Biol. Ed. M.M. Gross, Plenum Press (in press). Novoa W.B., Winer, A.D., Glaid, A.J., Schwert, G.W. (1959) J. Biol. Chem. 1143-l 148. 234:

965

BIOCHEMICAL

Vol. 63, No. 4,1975

22.

23. 24.

Et-win, Rawat, Veloso

V.G., A.K., D.,

Tabakoff, Kuriyama, Passonneau,

B., K., J.V.

Theorell,

H.

AND BIOPHYSICAL

and Bronough, R. (1971) and Mose, J. (1973) J. and Veech, R.L. (1972)

RESEARCH COMMUNICATIONS

Molec. Pharm. Neurochem. 20: J. Neurochem.

7: 23-99. 19:

2679-2686. 25. 26. 27.

Markovic, O., 195-205. Jornvall, H. Eds. Thurman, Doherty, J.D.,

and

Rao,

S.

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