Purification, properties and reconstitutive activity of a DPNH dehydrogenase

BIOCHEMICAL

Vol. 49, No. 5,1972

PURIFICATION,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROPERTIES AND RECONSTITUTIVE

ACTIVITY

OF A DPNB DEHYDROCENASE

Robert

F. Baugh and Tsoo E. King Department of Chemistry State University of New York Albany, New York 12222

Received

October

lo,1972

SUMMARY--A DPNB dehydrogenase has been solubilized from cardiac submitochondrial particles using Triton X-100 and purified by (NH4)2S04 fractionation, chromatography and density gradient centrifugation. It is an iron-flavoprotein free of lipid; the ratio of FMN:Fe:S was 1:28:28. The dehydrogenase can use CoQ as electron acceptor and the reaction is inhibited by Amytal and rotenone. The purified enzyme reacts with the cytochrome b-c particle to reconstitute antimycin A sensitive DPNB cytochrome c reducta;eT' At least chondria

(cf. -

stitutively With

8 soluble

1, 2) have been inactive--i.e.,

one exception,

The one which that

is

as rotenone

active

submitochondrial

m

venom at 30" In our

dehydrogenase

in the

(3).

This

with

all

inactive

system."

such inhibitors

dehydrogenase

with

(CoQ). from

of the DPNH dehydrogenase

by incubation

recon-

different

toward

reactive

mito-

to ubiquinone

shows behavior

ubiquinone

are

the "cytochrome

particles

product

particles

has been solubilized

phospholipase

A or a

(4).

since

effort

1958 with

up to a ferricyanide

similar

ubiquinone

from cardiac

literature;

are also

and submitochondrial

continuous

DPNB dehydrogenase

in the

preparations

to be a degradation

from

preparations

do not react

towards

and Amytal

considered

reported

they

these

in mitochondria

acceptor,

DPNE dehydrogenase

(S), Triton

activity

and shows inhibition to mitochondria reconstitution

EXPERIMENTAL--The

to search

for

we have recently X-100. of over

succeeded

The dehydrogenase 1700,

of antimycin Keilin-Hartree

toward particles.

A sensitive preparation

1165

active in solubilizing

has been

can use ubiquinone

characteristics

and submitochondrial

Copyright 0 1972 by Academic Press, Inc. AlI rights of reproduction in any form reserved.

a reconstitutively

purified

as an electron

rotenone It

a

and Amytal is

DPNB-cytochrome was prepared

also

active

c reductase. from bovine

Vol. 49, No. 5, 1972

heart

BIOCHEMICAL

according

to Method

of DPNH dehydrogenase other iron

acceptors (ll),

mined

toward

(10)

labile

were

pH 5.6 precipitation.

ferricyanide

(12),

(7), by the

and acid

described.

Other

CoQ (8),

published

The activities quinones

methods.

extractable

and

The non-heme

PMN (13)

operations

(9),

were

are detailed

deter-

in the

legends

figures. RESULTS--Solubilization

operations Hartree

were

at 30"

centrifugation 0.5% Triton

x g for

30 minutes,

x g.

sulfate

the presence

for

2% cholate,

fraction

toward

to about

was collected

20% to 62% sucrose 24 hours,

from

with

0 and 0.40

the column,

The fraction as before.

on 11 ml solutions

were

punctured

1166

in After

2 hours

ammonium

collected

between

fractionated

with was dissolved

was chromatographed of 1500 ml.

The

the highest

either

by ammonium sulfate

cell

over

the highest

This

pH 10.

then

for

saturation

fraction,

of an exponential

in 50 mM glycine,

to 8.5.

After

specific

an XM-50 membrane

on an agarose

with

After

showed

in an Amicon chromatographed

to 5'.

with

a bed volume

was concentrated

(430 ml)

was suspended

solution

which

to

25 ml of 50 mM glycine

was again

A-5m) with

further

the tubes

pellet

was centrifuged

The yellow-amber

and concentrated

was layered

the

cooled

The precipitate

between

(Rio-Rad

of 200 ml.

immediately

extracted

or by ultrafiltration 5 ml and then

The mixture

was fractionated

The extract

ferricyanide,

of a bed volume

x g for

pH 9.

of agarose

precipitation

was twice

same buffer.

protein

activity

supernatant

The fraction

in 25 ml of the

per ml was adjusted

X-100.

the mixture

of 0.5% cholate.

saturation

The Keilin-

of 430 ml and the pH adjusted

120 minutes

The yellow

ammonium sulfate.

second

of Triton

at 170,000

volume

indicated.

20 mg protein

and then

at 170,000

on a column

otherwise

5 minutes

at 30'

containing

about

--of the Dehydrogenase--All

for

incubation

and 0.44

unless

of 0.5% (v/v)

to a final

in

at O-4"

containing

in the presence

was incubated

each,

and Purification

conducted

preparation

pH 8.5

0.25

without

determined

sulfide

as previously

of the

1 (6)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(A-15m)

specific

column activity

in 0.5 ml aliquots density

centrifugation

and the fractions

gradient

of

at 283,000 with

the

BIOCHEMICAL

Vol. 49, No. 5, 1972

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Summary of Solubilization

I.

and Purification Specific Activity

Protein

Step

of DPNR Dehydrogenase* Total Activity

Yield

28

246,120

100%

940

201

188,940

77

275

360

99,000

40

229

382

87,480

36

120

605

72,600

30

A-15m agarose

91

743

67,610

27

Density

32

1720

55,000

22

Keilin-Hartree

preparation

8790 mg

Supernatant .25 - .44

(NH~>~SO~ fraction

0 - .40 (NH4)2s04

fraction

A-5m agarose

Gradient

*The activity is specific activity

in is

highest

ferricyanide

through

a Sephadex

is

given

the unit activity

activity

in Table

genase acid

acid

--of the Dehydrogenase

11 with

and Complex

extractable

1:28:28.

forms

(13).

of the purified

was removed

by passage preparation

and activity

whereas

the

iron

(14).

and acid

presented

jointly

with

sulfide

fluorometry

the typical

contained

in a ratio

of

and controlled

of the oxidized

and reduced

characteristics

here). Dr.

DPNR dehydro-

The dehydrogenase

labile

enzyme showed

enzyme are

of an

Preliminary

D. DerVartanian

from

results

revealed

4 DPNH

centers.

The dehydrogenase content

--et al.

and activities

A solubilized

by differential

not

composition

of the purified

spectra

conducted

iron-sulfur

below)

The absorption

(spectra

RPR experiments

--The

the phospholipase

I of Hatefi

FMN, non-heme

iron-flavoprotein

reactive

both

No FAD was detected

hydrolysis

Sucrose

at 30" and

A summary of a representative

and COG (see also

in Table (4)

pooled.

per minute

I.

ferricyanide

compared

were

G-25 column.

Some Properties toward

of umoles DPNR oxidized per mg protein.

differs toward

dehydrogenase

from Complex

ferricyanide. contains

negligible

1167

I,

inter

Complex lipid.

m,

both

I has about

by lipid 20-30%

The ferricyanide

lipid,

BIOCHEMICAL

Vol. 49, No. 5, 1972

Table

II.

Composition

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Activity

of DPNH Dehydrogenase Triton enzyme

Preparations

Venom enzyme*

(4,

7)

Complex

I (2)

Composition FMN, nmolesfmg

protein

Nonheme iron,

natomslmg

Labile

sulfide,

Lipid,

%

Activity,

1.13 protein

nmoles/mg

pmoles

protein

DPNH x min -1

1.12

1.4 - 1.5

32

19.6

26

32

31.1

25

0

0

22

-1 x ma

Ferricyanide**

1700

CoQ

(30°)

1500

16 (CoQ6)

(30°)

105

0

(38')

~14 (CoQ,)

*The venom enzyme refers to the DPNH dehydrogenase solubilized from submitochondrial particles by incubation at 30" with Naja naja venom or phospholipase A (4). **The ferricyanide activity for Triton enzyme and venom enzyme is in Vmax (acceptor) whereas that for Complex I is presumably determined at a fixed concentration of the acceptor.

activity

of the purified

-1 protein mg

at 30'

COQ Activity specificity genase

is

shows high

effectively

also

*K inhibition r&rired for

of Complex

I is

acceptors

(3)

1.

inhibited

for

III.

based

1700 pmoles less

low

the ubiquinone

preparation

for

although

considered

1168

that

activities.

of our dehydrogenase

of 0.22

with

than

the latter

ferricyanide

activity

Ki*

the dehydrogenase

may be also constant, 50% inhibition.

DPNH dehydro-

the dehydrogenase

with

x

200 at 38'.

is much higher

FMN content;

and very

-1

--of DPNH Dehydrogenase--The

The CoQ activity

The inhibition

than

DPNH x min

the Triton-solubilised

on the

menadione

inhibited

the Keilin-Hartree

Kotenone

about

Characteristics

in Table

enzyme

as shown in Fig.

that

is

-and Inhibition

summarized

preparation

for

while

of electron

of the Sanadi

Amytal

dehydrogenase

was similar

and 0.78

to that

mI4, respectively.

CoQ as an acceptor. as the amount

The Ki

of inhibitor

BIOCHEMICAL

Vol. 49, No. 5,1972

Table

III.

The Activity Acceptors*

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of DPNH Dehydrogenase

Toward

Acceptor

Various

Electron

Specific

Km bW

Activity*

Ferricyanide

5.0

1700

Jagalone

0.3

64

2,3-Dicyano-5,6-dichloro-1,4-benzoquinone

0.7

58

Cytochrome

0.16

5

Duroquinone

1.5

Menadione

3.8

.07

2,6-Dichlorophenolindophenol

2.9

0.2

2.4

COQ6

**

16

CoQ2

**

37

4.3

59

CoQl

x min -1 xmg -1 at

*Specific activity is in the unit of umoles DPNH oxidized 30“ except for CoQ homologs which were assayed at 22'. **It may not be meaningful to describe K for insolubility of these acceptors in an a&eous

was found

to be 1.75

respectively,

for

90% inhibition genase. of the the

was achieved

The Ki value inhibitor

nmoles

of rotenone

the dehydrogenase

for

at less

and the Keilin-Hartree

preparation.

than

per mg of dehydro-

4 nmoles

the dehydrogenase

particles

due to non-specific inhibition

binding

toward

in mitochondrial

preparations

of the

Sanadi

enzyme

(3)

in

higher

concentrations

of the

toward

the inhibitors

(3).

rotenone

corresponds

both

with

completely

two aspects: inhibitors

the latter for

1169

different

1.5 moles

are

mitochondrial

preparation

for

likely

components.

are similar from

A

Ki values

and rotenone

of the dehydrogenase but

to about

The higher

Amytal

of the inhibitors

characteristics

of the

per mg of protein,

per mole of FMN in the enzyme.

submitochondrial

These

and 8.25

CoQ and CoQ6 because sygtem.

to those

the behavior requires

Ki and shows a biphasic

behavior

Vol. 49, No. 5,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNfCATlONS

ImM

2mM

Amylol

Fig.

1.

Inhibition acceptor. particle,

of DPNH dehydrogenase HMP, the Keilin-Hartree from bovine heart.

The enzyme showed cytochrome

5 activity

very

similar

to that

previously

activity

toward

ferricyanide

p-hydroxymercuric temperature

induced

for

tution

region,

depended

The inclusion

cytochrome

affected

5.

We found

of the enzyme at 37",

the venom enzyme

However,

X-100

was ascertained

respiratory

incubation

for

at 0".

Less than

from DPNH to CoQ.

upon

CoQ6 as electron a submitochondrial

(15).

by incubation

an increase

The enzymic with

in the incubation

inhibition.

the Keilin-Hartree

inhibition

toward

was little

Reconstitution--Triton

tion

by Amytal with preparation,

activity

emerged reported

benzoate

of DPNH oxidase.

little

rapidly

3mM

Cmmotrotlon

Although it

0.0001%

to be a very

(1 ug per ml)

Triton

submitochondrial

preparation.

to be somewhere

in the region

cholate

also

was considerably

upon the of cholate

was found

removal during

of these

showed less

1170

inhibitor

showed

50% inhibi-

The locus of electron

inhibition

potent.

detergents

ammonium sulfate

potent

in the Successful

of the transfer

same reconsti-

as much as possible. fractionation

and column

BIOCHEMICAL

Vol. 49, No. 5, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3

5 E i% cl

2

I

I 1 1 lQQ2QQ3QQ4QQ5QQ6QQ

0

pg

OPNH

I

I

I

Lhydmgmsr

in proincubation mixture Fig.

2.

Reconstitution of DPNH cytochrome 2 reductase with DPNH dehgdroThe systems contained 2.4 mg of the cytochrome genase. --b-cl amounts of DPNH particle (17), 85 nmoles CoQ6, and varying dehydrogenase as indicted in a total volume of 0.5 ml of 50 mM phosphate buffer, pH 7.4. It was incubated at 0" for 1 hour. In assay, suitable aliquots of each mixture were diluted to a final volume of 3 ml of 0.1 mM cytochrome 2 and 30 mM phosphate buffer. Temperature, 22'.

chromatography

allowed

removal

was subsequently

removed

centrifugation.

A convenient

examine

with

the dehydrogenase

in submitochondrial

sample

particles

(16).

by our method Complex

taminated experiment A sensitive;

with

III

is given

for for

(the

with

confirmation

2.

6 ug of the antibiotic

1171

III

b-cl --

is

as it

example).

particle

was heavily

was found

to

activity

for

of the Green

c reductases.

The reconstitution

and gradient

of DPNH oxidase preparation,

of Complex

Cholate

of Triton

the cytochrome

and DPNH cytochrome

in Fig

not more than

for

chromatography

the contamination

Keilin-Hartree

reacted

was used only

column

dehydrogenase.

any inhibition

or in the form

succinate

from the

additional test

Our DPNH dehydrogenase prepared

of Triton

school con-

A representative to be antimycin

per mg of the dehydro-

Vol. 49, No. 5, 1972

genase

BIOCHEMICAL

inhibited

more than

From the figure, minute

one can obtain

DPNH oxidized

be considered

a value

since

of residual

of the activity

a many-fold

stimulation

serum

albumin

in

degree

of the

fresh

preparations

shown

in Fig.

detergent

cytochrome

2, might

soluble

showed

the dehydrogenase

system

a specific protein

activity

degraded

product

the introduction

dehydrogenase

20).

points

the pre-extraction

gel.

from

soluble

must opti-

We have also

observed

2 activity factors

by the however,

The decrease

dehydrogenase,

an increase

as

of the residual

it

a limiting

dehydrogenase

is similar

amount

was easily

of the

separated

to the reconstituted

The DPNH dehydrogenase

by

succinate

in the super-

activity.

was first

used by Xanuiga submitochondrial

resisted 200 pmoles This

with

further

20) to obtain

particles.

purification

DPNH oxidized

preparation

(19,

However,

after

reaching

by ferricyanide

has been considered

per mg to be a

of the venom enzyme (4).

The critical

agarose

obtained

(19,

far

by albumin.

factors;

ether-pretreated

of about

per minute

affected

reacted

(18).

X-100

thus

value

one.

aspect

from

This

of

by a

Among other

of the

the dehydrogenase

DPNH dehydrogenase

number

upon the age of the preparation;

the excess

DISCUSSION--Triton

per

as witnessed

or DPNH cytochrome

be due to several

particle,

only

exist

preparation.

dehydrogenase

5 reductase

apparently

serum albumin.

concentrations

in this

still

significantly

be a major

centrifugation;

natant

not

at high

--b-cl

cytochrome

muscle

DPN?I oxidized to a turnover

were

might

depended

were

When the

the conditions

of DPNH oxidase

stimulation

could

equivalent

c activity.

per mole of FMN at 22'.

by bovine

the heart

in the activity

is

detergent

stimulation

cytochrome

of 1.3 umoles

which

per minute

a minimum,

mum and traces

are

90% of the reconstituted

per mg of dehydrogenase,

1200 moles

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the present

of the submitochondrial

of cholate Actually, is

purification

the e

in purification,

the

removal

particles

with

and chromatography

of Triton

to the success

of the enzyme described

of the

1172

X-100

from

reconstitution.

Triton

X-100,

on the

the solubilized

BIOCHEMICAL

Vol. 49, No. 5, 1972

ACKNOWLEDGMENTS--Sufficient kindly it

donated

would

work

by Dr.

Karl

have been impossible

was supported

U. S. Public

Health

by grants

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

amounts

Folker,

directly

to finish from

of various

,this

the National

homologs

and indirectly. series

of CoQ were Without

of experiments.

Science

Foundation

them This

and the

Service. References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20.

J., Jr., Hegdekar, B. M., Kuboyama, King, T. E., Howard, R. L., Kettman, E. A., in E. C. Slater (Ed.), Flavins K. S., and Possehl, M-3 Nickel, and Flavoproteins, Elsevier, Amsterdam, 1966, p. 441. Huennekens, F. M., and Mackler, B., in T. E. King and M. Klingenberg (Fds.), Electron and Energy Transfer in Biological Systems, Marcel Dekker Inc., New York, 1971, p. 135. Sanadi, D. R., Huang, P. C., and Pharo, R. L., in T. E. King and M. Klingenberg (Eds.), Electron and Energy Transfer in Biological Systems, Marcel Dekker Inc., New York, 1971, p. 159. Salach, J., Singer, T. P., and Bader, P., J. Biol. Chem., 242, 4555 see also Singer, T. P., and Gutman, M., in Advances in (1967); Enzymology, 34, 79 (1971). Redfearn, E. R., and King, T. E., Nature, 2&, 1313 (1964). King, T. E., in Methods in Enzymology, 2, 202 (1967). Ringler, R. L., Minakami, S., and Singer, T. P., J. Biol. Chem., 238, 801 (1963); also Lusty, C. J., Machinist, J. M., and Singer, T. P., J. Biol. Chem., 240, 1804 (1965). Sanadi, D. R., Pharo, R. L., and Sordahl, L. A., in Methods in Enzymology, lQ, 297 (1967). Rusicka, F. J., and Crane, F. L., Biochem. Biophys. Res. Commun., 38, 249 (1970). King, T. E., and Howard, R. L., J. Biol. Chem., 237, 1686 (1962). K. S., and Jensen, D. R., J. Biol. Chem., 239, King, T. E., Nickel, 1989 (1964). King, T. E., and Morris, R., in Methods in Enzymology, l& 634 (1967). King, T. E., Howard, R. L., Wilson, D. F., and Li, J. C. R., J. Biol. Chem., 237, 2941 (1962); also Wilson, D. F., and King, T. E., J. Biol. Chem., 239, 2683 (1964). A. G., and Griffiths, Hatefi, Y., Haavik, D. E., J. Biol. Chem., 237, 1676 (1962); also Green, D. E., in T. E. King, H. S. Mason, and M. Morrison (Eds.), Oxidases and Related Redox Systems, Vol. 2, J. Wiley and Sons, New York, 1965, p. 1032. King, T. E., and Howard, R. L., Biochim. Biophys. Acta, 2, 489 (1962). Rieske, J. S., in Methods in Enzymology, lo, 239 (1967); also Green, D. E ., and MacLennan, D. H., in D. M. Greenberg (Ed.), Metabolic Pathways, Vol. 1, Academic Press, New York, 1967, p. 48. King, T. E., and Takemori, S., J. Biol. Chem., 239, 3559 (1964). King, T. E., in Advances in Enzymology, 8, 155 (1966). Kanuiga, Z., and Gardas, A., Biochim. Biophys. Acta, I&, 647 (1967). Kanuiga, Z., in H. Kamin (Ed.), Flavins and Flavoproteins, University Park Press, Baltimore, Md., 1971, p. 649.

1173