Respiration-linked phosphorylation in mitochondria of guinea-pig brown fat

Vol. 32, No. 3, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESPIRATION-LINKED

PHOSPHORYLATION

OF GUINEA-PIG J.I.

Pedersen,

E.N.

with

the

for

BROWN FAT

Christiansen,

technical

W. Delingsby Institute

Nutrition

IN MITOCHONDRIA

and H.J.

assistance

Grav

of

and R. Fabricius Research,

University

of Oslo,

NORWAY.

Received June 27, 1968 Uncoupling posed

as the

adipose

cause

tissue

of oxidative

phosphorylation

of the

thermogenic

(brown

great

usual

et al.

In contrast,

oxidative must

(Guillory

that

be emphasized

varying

possibility

that

phosphorylation suggested

It

1968a,b)

mitochondria

interpreted

as

et al.

1968).

1968;Hohorst have

out

fashion.

results

at different

evidence

to carry

normal

conflicting

and

produced

are able

an apparently

species

derive

ages

It from

and under

conditions.

consideration brown

fat

(Kornacker that

sites

these

several

experimental Serious

in

fat

1966;Thomson

such mitochondria

that

with

of brown

by some workers

and Racker,

et al.

phosphorylation

experiments

usual

et al.

1967;Prusiner

suggests

have been

phosphorylation

1967;Smith

others

Stratmann,

by brown

conditions

substrate-linked

(Lindberg

which

exhibited

experimental

representing

capacity

fat).

The low P:O ratios under

has been pro-

ATP generation

(Aldridge

has recently mitochondria and Ball,

was considered

carry

at only

given

to the

defective

19681,and

occurs

and Street,

been

it

oxidative

has been

one of the

three

1968).

important

492

therefore

to

investigate

the

Vol. 32, No. 3, 1968

details

of

BIOCHEMICAL

this

phorylation

presumed defective

and its

METHODS: than

24 hours'

homogenized

possible

old)

in a medium consisting

pestle

in a polypropylene

centrifuged

(1:3

at

was repeated

at

mitochondrial

w/v

dilution),

fraction

manipulations

using

to

Lindberg

and Ernster

tuted

isobutanol-benzene,

and the resulting medium.

0-4'C. by manometry

described

in the

or

legend

to

was measured essentially

this

n-Hexanol

was substi-

has been shown more

the phosphomolybdate complex (Hagihara 32 The non-extracted P was estimated by counting

1960).

according

to

determined system

scintillation

Lowry

et al.

by manometry

(Yellow

Springs

Mitochondria described

above

brown fat

Respiratory

or by using

a Clark

Instruments

with

Biological brains

brown fat

as controls

exhibited

Protein

(1951).

of guinea-pig

along

and carried

counter.

low,

oxygen

but

493

ratios

were

Oxygen Monitor).

mitochondria

significant

in

electrode

were prepared

Mitochondria

and

was determined

control

in the subsequent

RESULTS AND DISCUSSION: pig

were

in extracting

a Pack(ard Tricarb

animals

supernatants

40.31,

(1956).

since

was

and the procedure

was determined

of phosphate

according

Lardy,

at

teflon

in the homogenization

the procedures

Incorporation

SS-34)

rotor

was suspended

phosphorylation

spectrophotometry

effective

(Spinco

mM HEPES

The homogenate

The combined

were performed

Oxidative

for

rotor

(less

was gently

a loose-fitting tube.

(3000 g.min.).

of new-born

0.33 M sucrose-l

with

centrifuge

32000 g.min

tissue

guinea-pigs

of

4500 g.min.(Sorvall

once

centrifuged

I.

old

phos-

significance.

brown fat

and 3 weeks'

pH 7.4

Table

respiration-linked

physiological

Interscapular

buffer

All

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

from

as the

same

experiments.

new-born

phosphorylation

guinea

E

0 ttt 2

02

78.6

87.2 - 85.4

- 85.9 0.04

0.02

0.20 0.08

0.47 0.24 0.19

- 0.03

- 0.03

- 0.20 + 0.03

_+ 0.16 t 0.05 - 0.18

P:O

(6)

(8) (4)

32.5

132.0

14.5 36.6

35.4 13.5 10.5

_+ 6.1

- 30.8 - 12.5

Specific oxidation

(3)

Brain

of brown

112.0 -125.0 0.04 - 0.04 53.2 4.824.81 0.37 - 0.37 3.5 5.48-+ 0.12 0.03 _+ 0.006 (3) 2.77 - 2.86 as equivalents t muatoms oxygen/mg protein/min. or oxidation of Fe(CNj6 3- calculated ttt No cytochrome c added tt In the presence of 0.1 mM CoA and 1 mM DL-carnitine. When more than two experiments are presented the results are given reaction medium. The number in parentheses indicate the number of experiments.

Succinate

'T!?P~-~-+ antimycin Ascorbate-TMPD+02ttt Pyruvate-malate+Fe(CN)zSuccinate+Fe(CN)g-

TMPD ---T-----+ antimycin

- 82.3 + 15.5

82.0 105.6

Succlnate

+ 11.7 + 4.5 - 15.8

58.8 18.0 15.2

Brown fat

and P:O ratios observed with mitochondria and brain of new-born guinea-pigs

Pyruvate-malate+ DL-B-hydroxybutyrate+02 DL-B-hydroxybutyrate+02tt DL-B-hydroxybutyrylL-carnitine+02 Succinate+02

rates

Specific oxidation

Oxidation

Reaction

TABLE I

- 0.26

_+ 0.23

- 1.97 - 2.02

P:O

to 0 (2e). to the as mean f s.d.

0.72 1.00 0.36

0.48

0.37

1.60 1.53

2.56 1.60 2.34

fat

Vol. 32, No.. 3, 1968

under with

the

BIOCHEMICAL

prevailing

conditions

pyruvate-malate

and respiratory

between

hydroxybutyrate linked.

is

phosphorylation (Borgstrbm

et al.

oxidation nearly with

with identical

brain

to the

1955).

butyryl-L-carnitine

part

assumed

linked

fat

+ III).

and that

obtained

with

DL-6-

at

least

is respiratory

there

is no substrate

oxidation

P:O ratio;

this

chain-

of DL-6-hydroxybutyrate

to the

mitochondria,

substrateI +I1

that

Compared

both

found

(sites

showed considerably brown

The P:O ratio

may represent

figure

that

generally

I>.

phosphorylation

this

suggests

It

(Table

as substrate chain-linked

The difference

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

free (6-fold)

but

contrasts

acid,

enhanced

accompanied with

DL-6-hydroxy-

the

by a results

mitochondria.

Manometric measurements were carried out at 30°C in a medium cjvtaining 10 mM potassium phosphate buffer,pH 7.4, with 333 rnpc P/ *; 5 mM MgC12; 60 mM HEPES (Calbiochem) buffer, pH 7.4; ?::/f,fs b ovine serum albumin (Mann Research, fatty acid-poor); 220 mM sucrose; 30 UM cytochrome c; 2 mM ATP; 50 mM glucose; 11 units/ml of hexokinase (Sigma), in a total volume of 2.0 ml. Substrates were used at the following concentrations: 5 mM pyruvate and 5 mM malate in the presence of 0.25 mM NAD; 10 mM DL-B-hydroxybutyrate; 6 mM DL-B-hydroxybutyryl-L-carnitine; 10 mM succinate 20 mM ascorbate (+ 0.6 UM rotenone). The (+ 0.6 UM rotenone); TMPD concentration was 0.4 mM, antimycin being used as 1 ug/mg of mitochondrial protein. Mitochondrial protein was 4-8 mg/vessel and the respiration was measured for 15-20 minutes. The ascorbate/ TMPD system for site III phosphorylation was essentially that of Sanadi and Jacobs (19671, and the succinate/antimycin/TMPD system for the same site was that of Lee et al. (1967) adapted to manometry. The ferricyanide method was modified after Schatz and These reactions were carried out at 30°C in a Racker (1966). The incubation medium and substrate total volume of 3.0 ml. concentrations were as above except that cytochrome c was omitted, the sucrose concentration was 190 mM, and that 1.5 mM KCN was added. After 5 minutes of preincubation, the reaction was started by the addition of K3Fe(CN)6 to a final concentration of 0.8 mM. Mitochondrial protein was 2 mg/cuvette and the reduction of ferricyanide was followed spectrophotometrically for 5 minutes. correction was made for the nonIn calculating the P:2e ratio, phospborylating antimycin-insensitive reduction of the ferricyanide (which amounted to approximately 20% of the reduction found in the absence of antimycin).

495

Vol. 32, No. 3, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Further fat

analysis

of the phosphorylation

mitochondria

revealed

while

II

sites, Thus all

of

significant

sites

the following

(site

II);

by TMPD over

results

further

II (site

III);

of the respiratory

sites

II

and III.

by insuccinate succinate+02

site

the measurable

three

affected.

+ III);

the antimycin-inhibited

part

to all

were accompanied

Succinate+02(sites

that

of brown

were the more strongly

reactions

suggest

between

may apply

ascorbate-TMPD-+02

shunted

to the terminal

uncoupling

and III

P:O ratios:

+Fe(CN)z-

buted

that

sites

(site

III).

The

phosphorylation

chain

is

linked

equally

distri-

Endogenous respiration

was not

detected. Several Guillory

investigators

and Racker,

serum albumin

on the

mitochondria. 1% bovine

rates

with

the presence to Guillory

of 1% serum albumin

to improved

in the

stimulation In contrast

mitochondria, oxidative

rates, with

However,

proved

not

presence in the

EDTA (l-2 effect.

although to affect or

on the presence accompanied

of

by a

rate. in new-born

guinea-pig

of 3 weeks'

old animals

revealed

(Table

II).

,

according

situation

496

mM) in

Moreover

media, appear

or

of phosphorylation

medium, again

phosphorylation

of

the pyruvate-malate+

dependent

of the respiratory

those

incubation did

of

on P:O ratios

defatted

detection

absolutely

incubation

to the

effect

substrates.

in the

the acid-poor)

was without

associated

reactions.

ferricyanide

normal

fatty

(extensively

respiratory

the P:O ratios

serum albumin

fat

(1968))

effects

of brown fat

conditions,

of

medium likewise

and Racker

succinate+02

3-fold

capacity

(Mann Research,

a variety

1968;

the benevolent

medium had no demonstrable

the homogenization

with

have stressed

phosphorylating

serum albumin

respiration

strongly

and Street,

Under our experimental

homogenization

leading

1968)

(Aldridge

This

striking

brown near

Vol. 32, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II -Oxidation

rates

and P:O ratios

mitochondria

of 3 weeks'

Reaction

Specific

Pyruvate-malate+ Succinate+02

Succin,ate-TMP~---~-~O antlmycln 2 Ascorbate-TMPD+02

Experimental

difference

conditions

In fact,

brown fat

cells

propor-tion

suggests linked the

of

of fat,

be expected animals.

Contrary the

II

ascorbate-TMPD+02 the brown fat

(3)

2.60

* 0.61

120.3

* 19.9

(3)

0.93

* 0.04

51.5

*

1.0

(3)

0.05

* 0.01

159.1

f

3.3

(3)

0.05

* 0.01

in the

old animals

procedural

or free

to this

to Table

of normal

mitochondria at

P:O ratio

of the older

animals

I

(the

mitochondria

497

II

capacity difference

of

reveals

respiratory

that

difference

reactions). remains

and

possess fully

P:O ratio

and ascorbate-TMPD+02 identical

strongly

or succinate+02 Table

site

would

of older

evidence

Similarly,

and succinate+02

in these

uncoupling

phosphorylation

by the TMPD and succinate----V---z-+02 antimycln reactions).

acid

the

since

a much higher

in the mitochondria

expectation,

I.

signifi-

conditions,

contain fatty

(as represented

TMPD (succinate----t---7-+02 antlmycln Low, and nearly

legend

experimental

3 weeks'

the malate-pyruvate+02

P:O

* 21.2

identical

phosphorylation

phosphorylation

oxidation

a phenomenon of physiological

establishment

to site

brown fat

54.5

to be more prominent

succinate+02

coupled

under

rate

with

old guinea-pigs

as described

may represent

cance.

observed

Site

virtually

of III

uncoupled

reactions). control

ratios

were

Vol. 32, No. 3, 1968

exhibited

by mitochondria

guinea-pigs control

BIOCHEMICAL

(Table while

proceeded

the

with

from

III). oxidation

The following

albumin

to the

homogenization

Respiratory

and ascorbate-TMPD ADP control.

failed

to elicit

(Hohorst

TABLE III ratios t of brown

control

additional of serum

and Rafael,

fat

1968).

mitochondria

Manometry tt new-born 3 wks old

Substrates

to the

GTP, and additions

medium

old

showed clear

or additions

media

EDTA, ATP,

and 3 weeks'

oxidation

measurable

pretreatments

control:

new-born

of succinate

or incubation

respiratory

both

Pyruvate-malate

no or barely

homogenization

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Polarography ttt new-born 3 wks old

Pyruvate-malate

2.6

2.5

1.2

1.3

Succinate

1.1

1.1

1.0

1.05

1.0

1.0

Ascorbate-TMPD t Respiratory

control

ratio

tt The protein concentration ttt Protein concentrations born and 3 weeks' old

=

extramitochondrial

normal

of

guinea-pig

characteristics

The observation respiration-linked

with ADP without ADP

described in the legend system was omitted.

of 0.2 mM Ca 2+ to mitochondria free 2+ Ca failed to stimulate respiration.

addition

Mitochondria

rate rate

was 2 mg/ml; 2 mM ADP; 30°C. were 0.5 mg/ml and 0.2 mg/ml for newanimals, respectively; 0.1 ti ADP; 25'C.

The incubation medium was that except that the glucose-hexokinase Similarly,

respiratory respiratory

that

brown of states

the

fat

therefore

do not

3 and 4 oxidations

establishme.nt

phosphorylation

of near in animals

498

to Table

of

exhibit (Chance,

1959).

normal aged

I,

3 weeks was

Vol. 32, No. 3, 1968

not

8lOCHEMlCAL

accompanied

presence

by improved

in brown

adipose

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

respiratory tissue

control

again

of a specialized

favours

type

the

of

mitochondrion. That

these

findings

mitochondria

is

ferricyanide

system

was effectively

our experimental

conditions),

under

a phenomenon condition

supported

were not

exhibited (Lee

mitochc'ndria

is

et al.

phosphorylation,

to

The striking old that

the

related the

brown

uncoupling to the

thermogenesis

by antimycin being

suggest

taken

are

of small

that

(80% to represent

in

the

brown

fat

capable

intact

found

mitochondria in

of coincident and partially

Part

chain-linked

of this

phosphorylation

to sites

in phosphorylation fat

of respiration-

magnitude.

and insignificantly

physiological

implications

escaped

I,

succinate-

by mitochondria

guinea-pigs though

increase

guinea-pig

this

The respiratory site

the

blocked

our results

substrate-linked.

that

to damaged

1967).

of new-born

is coupled

fact

exclusively

In conclusion,

linked

by the

attributable

capacity

II

and III.

of

3 weeks'

justify

the

new-born

animals

may have

significance

function

of this

tissue.

Moreover,

existence uncoupled

in brown phosphorylation

assumption

fat

tissue have

of not

our notice.

ACKNOWLEDGEMENT We gratefully acknowledge financial assistance from "A/S Freia Chocolade Fabriks arbeidsfond for ernmringsforsking". We also thank Dr. Jon Bremer for a generous gift of DL-8-hydroxybutyryl-L-carnitine. REFERENCES 1. 2.

Aldridge, BorgstrBm,

315.(1968). W.N., and Street, B.W., Biochem.J.107, B., Sudduth, H.C., and Lehninger, A.L., J.biol.Chem.215,571 (1955).

499

Vol. 32, No. 3, 1968

3.

4. 5. 6. 7.

8. 9.

10. 11. 12. 13.

14. 15. 16. 17.

Chance,

BIOCHEMICAL

B.,

in G.E.W. Wolstenholme and C.M. OConnor (Editors), Ciba Foundation Symposium on the Regulation of Cell Metabolism. J. and A. Churchill ltd., London, p. 91 (1959). Guillory, R.J., and-Racker, E., Biochim. biophys. Acta 153, 490 (1968). Hagihara, B., and Lardy, H.A., J.biol.Chem. 235, 889 (1960). Hohorst, H.-J., and Rafael, J., Hoppe-Seylers Z.physiol. Chem. 349, 268 (1968). Hohorst, H.-J., and Stratmann, D. 4th Meeting, Federation of European Biochemical Societies, Oslo, Abstr. 435 (1967). Kornacker, M.S., and Ball, E.G., J.biol.Chem. 243, 1638 (1968). Lee, C.-P., Sottocasa, G.L., and Ernster, L., in R.W. Estabrook and M.E. Pullman (Editors), Methods in Enzymology, Vol. X, Academic Press Inc., New York. p. 33 (1967). Lindberg, O., and Ernster, L., in D. Glick (Editor)? Methods of Biochemical Analysis, Vol. 3, Interscience Publishers Inc., New York. p. 1 (1956). Lindberg, O., de Pierre, J., Rylander, E., and Afzelius, B.A., J. Cell Biol. 34, 293 (1967). Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., J. biol. Chem. 193, 265 (1951). Prusiner, S.B., Eisenhardt, R.H., Rylander, E., and Lindberg, O., Biochem. biophys. Res. Commun. 30, 508 (1968a). Prusiner, S.B., Williamson, J.R., Chance, B., and Paddle, B.M., Arch. Biochem. Biophys. 123, 368 (1968). Sanadi, D.R., and Jacobs, E.E., in R.W. Estabrook and Pullman, M.E. (Editors), Methods in Enzymology, Vol. X, Academic Press Inc., p. 38 (1967). Schatz, G., and Racker, E., B&hem. biophys . Res. Commun., 22, 579 (1966). Science Smith, R.E., Roberts, J.C., and Hittelman, K.J., 3,

18.

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653

(1966).

Thomson, J.F., Smith, D.E., Nance, Comp. Biochem. Physiol.,

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S.L., 25,

and Habeck, 783 (1968).

D.A.,