Pyridine nucleotide transhydrogenase activity of soluble cardiac NADH dehydrogenase and particulate NADH-ubiquinone reductase

Vol. 60, No. 3, 1974

BIOCHEMICAL

PYRIDINE

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NUCLEOTIDE TRANSHYDROGENASE ACTIVITY

OF SOLUBLE

CARDIAC NADH DEHYDROGENASE AND PARTICULATE NADH-UBIQUINONE C. Ian Ragan*,

R. Widger

William

Department State

Received

August

and Tsoo E. King

of Chemistry

University

of New York

Albany,

REDUCTASE

New York

at Albany

12222

19,1974

SUMMARY--Pyridine nucleotide transhydrogenase activities of a highly purified soluble NADH dehydrogenase and particulate NADH-ubiquinone reductase (Complex I) differ in their pH opti !J$ (5.0 and 6.0, respectively) and in their sensitivity to inhibition by Mg and ATP. The oxidation of NADPH with ferricyanide as acceptor is very similar in both preparations with a pH optimum around 5.0. It is concluded that Complex I possesses two types of transhydrogenase activity, whereas only one has been found in the soluble dehydrogenase. Recently,

the

transhydrogenase after

nearly

troversy.

Hatefi

ubiquinone

reductase)

it

also

shows

considerable

From visible

interacted

with

site

was involved

have

with

been challenged

we compare

the

dehydrogenase

in the those

-c

Present

Copyright All rights

reported

they

reported

investigators of NADPH with

in this

laboratory

(8) were

obtained

reductase

at Vmax for

address:

Department Southampton,

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

894

con-

"Complex

I"

(NADH-

concluded (3)

that

NADPH

and proposed

by Ernster . (e.g. 6, 7).

-et -al.

Complex

and

transhydrogenase

Their

that

conclusions (4,

In this

5) and

report

a soluble

I and with

NADH

(8). Complex

and specific

of Physiology Southampton,

little

reaction.

of

ferricyanide

in-

of NADPH by ferricyanide

transhydrogenase

previously

vigorous

with

that

nucleotide

MATERIALS AND METHODS--Preparations

cyanide

but

2 of NADH dehydrogenase

interactions

NADH dehydrogenase

progress

oxidation

pyridine

by other

isolated

the

moderate 2) have

nucleotide

has come under

and EPR spectroscopy

EPR Center

are at variance

(1,

catalyzes

and pyridine

preparations

20 years

and Hanstein

activity.

this

of NADPH oxidation

in mitochondrial

vestigation,

that

subject

(1,

I (1,

9) and soluble

activities 8) were

assayed

and Biochemistry, England.

of NADH-ferrias previously University

of

Vol. 60, No. 3,1974

described.

BIOCHEMICAL

Pyridine

estimated

nucleotides

by the method

hydrogenase

activity

rically

at

(2) *

Variations

and table

AND BIOPHYSICAL

were

of Lowry

with

363 nm according in assay

Pyridine

indicated

la

shows the

In agreement

a broad

ferricyanide

pH optimum

oxidation

by ferricyanide

pH 5 (12).

corresponding

for

previous

(4),

especially

in the

and Hanstein

respective

the

2.8% of the

figure

same regardless

of

NADPH oxidation

were

nature

and about

The NADH-ferricyanide soluble

approximately

the

same.

APAD as acceptor. dehydrogenase.

The pH optimum of the

preparation.

However,

soluble

The optimum

enzyme has an activity

Abbreviations:

Figure

Id

the

is

was between only

oxidation

lb shows

the

but

of

Complex

activities

is

lower

at pH 8, the

one-tenth

of the

of this

the

the

pH dependence than

the

pH profiles --i.e.

optimum

of than

that

same optimum

and 6.5, maximal

APAD, acetylpyridineadeninedinucleotide; phenolindophenol.

I are

shown

one-third

was

Complex

I with

of

activity is

at which

the

NADH-

of

either

entirely

Unlike

DCIP,

soluble

for

pH the

activity.

in

of that

activity

found

of NADPH-APAD activity pH 6.0

rate

acceptor,

NADPH dehydrogenase

shows the

NADPH

of NADPH

lower

enzyme or NADH-ferricyanide pH profile

the

was approximately

The NADH-APAD activity

APAD activity

different.

while

for

NADH oxidation.

activities

activity

dehydrogenase,

the

NADH

showed

NADH oxidation;

electron

pH 8 for

soluble

optimum

considerably

of the

NADH and

oxidation

as acceptor,

The NADH- and NADPH-ferricyanide lc.

of

Figure

ferricyanide the

by the

the

to observe

activities

of

pH optima,

rate

at pH 7.5.

with

pH dependence

while

respective

failures

Both

activities

at pH 5 for

1

by Hatefi

findings,NADH

pH 8 (8),

At the

only

APAD as acceptor.

the

trans-

spectrophotomet-

catalyzed

previous

around

was still

may account

as acceptor with

centered

was about

NADPH oxidation

of

was

nucleotide

as adapted

are

Protein

legends.

dehydrogenase.

Fig.

Sigma.

was measured

(11)

procedures

with

were

(10).

to Kaplan

NADPH oxidation

with

from

as acceptor

RESULTS AND DISCUSSION--Figure

fact

purchased

--et al.

APADl

RESEARCH COMMUNICATIONS

soluble the

2,6-dichloro-

Vol. 60, No. 3, 1974

BIOCHEMICAI.

soluble

enzyme,

Complex

I was distinctly These

genase

facts

activity.

the

pH profile

that

at 5.0

cyanide

of similar

activity

not

found

appear

only

that

and appears

NADPH oxidation

with

APAD as acceptor

ferricyanide

I may have

two types

that

of

the

to be associated

pH dependence. I,

RESEARCH COMMUNICATIONS

with

resembles

in Complex

to catalyze

from Complex

One of these

has a pH optimum

activity,

of NADPH oxidation

different

suggest

AND BIOPHYSICAL

The other

has a pH optimum

of transhydrodehydrogenase, an NADPH-ferri-

transhydrogenase at

6.0

by ferricyanide.

a)

as acceptor,

soluble with

by

to 6.5

These

and does

properties

1. - MOl60-

OEHYDl?OSENA6E- 4.0

c

l-a-

: loo2 2 60-

5

6

PH

7

tf

60-

$

40-

*

20-

8 -

I6

! c

I4

E

lo

5

6

5

6

PH

7

8

7

s

12

5

6

ij

6

:

PH

Fig.

1.

Effect of pH on NADH- and NAHPH-acceptor activities of Complex I and soluble NADH dehydrogenase. NADH-ferricyanide (NADH-FeCN) activity was measured at 420 nm with 0.125 mM NADH, 1 mM K Fe(CN)6 and 5 ug of enzyme protein per ml. NADPH-ferricyanide (N Ai3PHFeCN) activity was measured at 420 nm with 0.5 mM NADPH, 1 mM K3Fe(CN)6 and 25 pg of enzyme protein per ml. NADH-APAD transhydrogenase activity was measured at 363 nm with 0.5 mM NADH, 1.0 mM APAD and 5 ug of enzyme protein per ml. NADPH-APAD transhydrogenase activity was measured at 363 nm with 0.5 mM NADPH, 1.0 mM APAD and 25 pg of enzyme protein per ml. The media were 0.1 M K-acetate (pH 5.0 and 5.5) or 0.1 M potassium phosphate (pH 6.0, 6.5, 7.0 and 8.0). Temperature was 38'. Appropriate Specific corrections were made for small non-enzymic activities. activities of the soluble NASH dehydrogenase and Complex I in the NADH-ferricyanide assay were 833 and 340 pmoles NADH/min/mg of protein, respectively, in terms of Vmax for ferricyanide at 30".

896

Vol. 60, No. 3,1974

BIOCHEMICAL

are reminiscent isolated

of

the mitochondrial

by Kaplan

(ll),

digitonin.

This

significant

to note

of the

the

that

this

than

that

however,

we also

find

to preparation

of the

I and that

Complex

similarity found

the mitochondrial soluble

of

ferricyanide

activity

Transhydrogenase

activity

the

soluble

the

two types

by Mg about

Mg

is

(2).

during

in the

In part,

the

prepa-

activity

from

to as low as 0.7

at high

in the

for

purified

both

enzymes

I to give

assayed that

of

Table

little

897

of

inhibition of Complex

was substantially

(4,

with

good separation

the

activity therefore,

dehydrogenase.

unaffected

activity

6) would

NADPH dehydrogenation soluble

at pH 5.5

NADPH-DCIP activity

I,

NADPH-

NADPH-DCIP activity

at pH 7.5,

considerable

I.

and was found

activity

Similarly,

pH 6.0,

of

of 10 mM MgSO4 or 1 mM ATP. was assayed

transhydrogenase

may be two pathways appears

at

of

I and the

in Table

transhydrogenase

reports

The data

Complex

dehydrogenase,

or ATP.

I,

pH, since

low pH.

soluble

activity some inhibitors

summarized

presence

the

2+

in Complex Previous

enzyme at

are

Complex

assayed

the mitochondrial only

one of which

while

dehydrogenase,

60% inhibited.

there

salts

on both

APAD as acceptor

In the

52% by either

or ATP, while

be correct

Id,

and Hanstein

transhydrogenase

tested

by the

with

of reaction.

soluble

of

(Triton

in Fig.

shown

at pH 5.5 with

affected

2+ or ATP was observed,

reaction

purification

I,

particles,

The results

and at pH 7.5 with

enzyme,

I was inhibited

the

were

was assayed

little

2+

the

It may be mentioned

occasionally

sin submitochondrial

transhydrogenase

to be relatively

the

I,

for

may be

alone).

variation

between

NADH dehydrogenase.

by Ms

used

bile

considerable

It

6.0.

NADH dehydrogenase

by Hatefi

inhibitory

by means of

of protein.

In view Complex

Complex

reported of

were

salts

of

of

soluble

I (bile

activity

removal

umoles/min/mg

that

the

due to

preparation

soluble

(digitonin),

is probably

ration;

of

detergents

transhydrogenase

purified

has a pH optimum

and Complex

greater

RESEARCH COMMUNICATIONS

nucleotide

has been partially

different

transhydrogenase

considerably

pyridine

transhydrogenase

and cholate),

that

which

transhydrogenase

X-100

AND BIOPHYSICAL

was is a partial

thus

appear

to

was found

with

the

aupport

the

view

in Complex

I,

only

I

ATP, 1 mM

2.48

2.53

Mgso4,

10 mM

3.04

2.45

2.52

None

1 mM

10 mM

2.70

18

16

9

7

%

Inhibition

6

0.21

0.21

0.45

0.82

0.98

1.00

umolesiminfmg

Rate

NADPH oxidation

NADH Dehydrogenase

with

52

52

18

2

%

Inhibition

APAD

indicated

and by Particulate

Rate

0.24

0.21

0.59

0.95

0.99

1.05

umoles/min/mg

acceptor

59

64

10

6

%

Inhibition

DCIP

NADH-Ubiquinone

Activities of the soluble dehydrogenase were assayed as follows: NADPH-K3Fe(CN)6 and NADPH-APAD activities of NADH dehydrogenase were assayed under the same conditions as described in the Legend of Fig. 1 at pH 5.5; NADPH-DCIP activity was determined spectrophotometrically at 600 run in 1 ml final volume containing 0.1 umole of K-acetate pH 6.0, 0.5 umole of NADPH, 50 nmoles of DCIP and 25 pg of enzyme protein at 38'. NADPH-K Fe(CN)6 and NADPH-APAD activities of Complex I were assayed as in Fig. 1 at pH 5.5 and pH 7.5, respectively. N&lPHDCIP activity of Complex I was determined as that of the dehydrogenase but at pH 7.5.

Complex

ATP,

MgS04,

None

Rate

K3Fe (W

by Soluble

umoleslminlmg

I").

Catalyzed

("Complex

Addition

Reductase

NADPH Oxidation

NADH

dehydrogenase

Soluble

TABLE I.

BIOCHEMICAL

Vol. 60, No. 3,1974

The results oxidation

presented

by Complex of

ation

stereospecificity

the

chondrial the

(see

Ref.

respiratory 15 for

examined

cyanide It

(14). specificity sults

the

soluble ment

for

under

conditions

In view

of

from

all

pH optima

16,

one soluble

GM 16767)

from

oxidation

17)

the

be obtained

through

consider-

involved,

in the

membrane

these

enzyme--

the

work

U. S. Public

Type

all

I and Type

preparations

4B specific

is

also

ferri-

been made on the

described.

stereo-

Definitive

beyond

II so

particles

re-

a reasonable

doubt

are due to cleavage

of

NADH dehydrogenase-containing

in Fig.

solubilizing

1 of Ref.

such

as the

activities

respiratory was supported Service,

of

the seg-

agents

17). reactivity,

(by deduction),

Health

14)

or

has yet

considerations,

and the

both

by different

and stereospecificity

the mito-

of NADH by oxygen

preparations

sites

since

of

atom of NADH (13,

for

have demonstrated

(as depicted

ACKNOWLEDGMENT--This

evidence

for

experiment

inner

activity,

Further

NADH dehydrognease

at different

transhydrogenase are

soluble

varying

behavior,

the

no direct

mitochondrial

particles.

is 4B specific

NADH dehydrogenase

enzymes

NADPH

4A-hydrogen

of submitochondrial

example

various

of the

terminology)

that

regarding

reactions the

RESEARCH COMMUNICATiONS

situation

NADH dehydrogenase

Likewise,

the

might

involves

presence

of

(see

that

the

is true

the

of the

chain

(14).

in the

clarify

NADPH dehydrogenation

transhydrogenase

while

far

here

I and submitochondrial

two pathways of

AND BIOPHYSICAL

we think

inhibition that

the

NADH and NADPH oxidation

chain-linked by the

NADH dehydrogenase. grants

National

(HL 12576 Institutes

and

of Health.

References 1. 2. 3. 4.

5. 6.

Hatefi, Y. (1973) Biochem. Biophys. Res. Commun., 50, 978. Hatefi, Y., and Hanstein, W. G. (1973) Biochemistry, I.& 3515. Orme-Johnson, N. R., Or-me-Johnson, W. H., Hansen, R. E. Beinert, H., and Hatefi, Y. (1971) Biochem. Biophys. Res. Commun., 44, 446. Ernater, L., Lee, C-P., and Torndale, U. B. (1969) in The Energy Level and Metabolic Control in Mitochondria (Papa, S., Tazr, J. M., Quagliariello, E., and Slater, E. C., eds.) p. 439, Adriatica Editrice, Bari. Rydstrsm, J., Teixeira da Cruz, A., and Ernster, L. (1971) Eur. J. Biochem., 22, 212. Rydstrtim, J., Holk, J. B., and Ernster, L. (1973) Biochim. Biophys. Acta, 305, 694.

899

Vol. 60, No. 3,1974

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

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Rydstriim, J. (1974) Eur. J. Biochem., 5, 67. Baugh, R. F., and King, T. E. (1973) Biochem. Biophys. Res. Commun., 2, 1165. Hatefi, Y., Haavik, A. G., and Griffiths, D. E. (1962) J. Biol. Chem., 237, 1676. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem., 193, 265. Kaplan, N. 0. (1967) 2 Methods in Enzymology. Vol. X (Estabrook, R. W., and Pullman, M. E., eds) p. 317, Academic Press, New York and London. King, T. E. and Baugh, R. F. (1974) Biochim. Biophys. Acta, in-press. Kawasaki, T., Satoh, K., and Kaplan, N. 0. (1964) Biochem. Biophys. Res. Commun., 17, 648. Ernster, L., Hoberman, H. D., Howard, R. L., King, T. E., Lee, C. P., Mackler, B., and Sottocasa, G. (1965) Nature, 207, 940. Slater, E. C. (1966) in Discussion of Flavins and Flavoproteins (Slater, E. C., ed.) p. 487, EGevier, Amsterdam and New York. J. Jr., Hegdekar, B. M., Kuboyama, M. King, T. E., Howard, R. L., Kettman, E. A. (1966) in Flavins and Flavoproteins M ., Nickel, K. S., and Possehl, (Slater, E. C., ed.) p. 441, Elsevier, Amsterzm and New York. King, T. E. (1966) in Discussion of Flavins and Flavoproteins (Slater, E. C., ed.) p. 480,%sevier, Amsterdam and New York.

900