Substrate dependent compartmentation of ATP in rat liver mitochondria

Vol. 13, No. 5, 1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMJNICATIONS

SUBSTRATE DEPENDENT COMPARTMENTATION OF ATP IN RAT LIVER MITOCHONDRIA. F.A.Hommes

and J.A.C.M.van

der Beek

Department of Biochemistry, University of Nijmegen, School of Medicine, Nijmegen, The Netherlands.

Received September 24, 1963 It

has been

amounts

reported

earlier

of hexokinase

liver

nucleotides, is reached

stimulate

rate

of oxygen

for

the existence

an indication maximal

rate

of oxygen

rate

limiting.

malonate

This

Klingenberg, pathways

maximal

investigate ate

(1948).

with

was apparently

or glutamate, uptake

an initial could

not

of dynamic

(Klingenberg,

1963), pyridine

dehydrogenases.

mitochondria

The extent

It

malate,

were

observed

stimulation be released

by

organization

has indicated nucleotides

was therefore

described

were

with

system

prepared

of pyridine

using

and Williams,

essentially

nucleotide

a Chance-Aminco

(Chance, 1954) at the wave length Oxygen consumptions were measured (Chance

as this

obtainable

of oxygen

theory

of the above

spectrophotometrically,

electrode

ATP,

as

of that

is

a

essential

of interest

to

@-hydroxy

butyr-

as described

by

in more detail.

Rat liver

Schneider

does not

interpreted

reaction

After

which

of mitochondrial of the

inhibition

rate

succinate

kinetics

proposed

in mitochondria

of reduction

as substrate Methods:

with

for

a

phosphorylation.

recently

conditions the

the

the hexokinase

was observed,

of oxidative in his

medium degree for

peculiar

below

until

has been

barrier

was used as substrate.

an inhibition

ADP or uncouplers hydrogen

Rather

butyrate

of respiration,

of a permeability

was observed

of externally

of hexokinase

phenomenon

was far

of increasing

absence

respiration,

addition

This

of ATP for

effect

as substrate.

when g-hydroxy

a further

uptake.

utilisation

addition

in the

in an increased

at which

added ADP. The availability

that

mitochondria

results

added adenine plateau level the

(Hommes, 1962)

to rat

pair 340-374 polarographically 1955).

reduction

double

Hexokinase

my

was monitored

beam spectrophotometer

(Chance and Williams, with a Clark oxygen was purchased

from

1956).

Boehringer

and Soehne. Results: hexokinase

The kinetics

of oxygen

to mitochondria

in

utilisation the presence

are

shown

of glucose

in fig.1.

Addition

and (3-hydroxy

butyrate

of

Vol. 13, No. 5, 1963 results

BIOCHEMICAL

in a slightly

respiration

is

increased

observed,

AND BIOPHYSICAL

respiration.

followed

RESEARCH COMMUNICATIONS

After

about

by an inhibited

4 min.

a burst

of

state.

Fig. 1. Kinetics of oxygen utilisation by rat liver mitochondria in the presence of glucose, S-hydroxy butyrate @-OH) and hexokinase. Protein concentration: 4.1 mg per ml. The numbers are the rate of oxygen consumption in mu moles 02 per min. The reaction was carried out in a medium containing 0.23 M sucrose, 10 mM K2HP04, 10 mM KCl, 5 mM MgC12 and 10 mM triethanol amine hydrochloride, pH 7.4 .

Fig.

2 shows

pyridine is

the corresponding

nucleotides

accompanied

oxidized

was measured

by a complete

in agreement

with

state

DPN linked

such inhibition

as substrate. inhibition

the

fig.

experiment

This

specific.

under

these

acceptor oxidation

of

nucleotides

that is

phase

nucleotides, a completely

inhibitory

for

that is

the

not

system

also

pyridine

only

nucleotide

possible

in

than

is

not

observed

with

glutamate,

malate,

linked

and there.

nucleotide

S-hydroxy

butyrate

as substrate

is also unimpaired as can be is the same as in the control

in these

acceptor,

ATP generated in turn

the presence

tightly

provided

compartmentalized

by which

butyrate DPNH is

the dehydrogenase. mitochondria

in

by the glucose-hexokinase

system that

via coupled

by oxidative indicates

of g-hydroxy

phosphorylation

the reduction

to the glucose-hexokinase ADP. This

with

of oxidative

process

of phosphate

is

observed

Succinate oxidation of oxygen utilisation

process

a faster is

were

conditions.

of pyridine

means that

available

This

1. The rate

Oxidation

presence

pyridine

pyridine

namely

phenomena

phenomena

seem to be rather

oxidation

of Klingenberg,

redox-state The inhibited

of mitochondrial

of the mitochondrial

fore

indicates

the

spectrophotometrically.

oxidation

the observation

malonate

oxidized

in which

dehydrogenases.

However,

seen in

experiment

phosphorylation in order

, at least

not

the system.

must be freely

to provide

ATP generated

This

the

in S-hydroxy

to such an extend

phosphate butyrate that

it

Vol. 13, No. 5, 1963 16mM 2.5 ml O.OSml

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

glucose

buffer M,

6.4mM

QOH I

03

mg

hexokinase

Fig. 2. Kinetics of oxidation and reduction of pyridine nucleotides in rat liver mitochondria in the presence of glucose, (3-hydroxy butyrate (S-OH) and hexokinase. Experimental conditions as in fig. 1. Protein concentration: 1.6 mg per ml.

controls

S-hydroxy

oxidation These ation

butyrate

of succinate differences

of the

oxidation,

or glutamate, may reveal

dehydrogenases

in

in contrast malate,

some unique the mitochondrial

to the ATP generated

characteristics

as to the organiz-

cristae.

References: Chance, B., Science 120, 767 Chance, B. and G.R.Wxiams, Chance, B. and G.R.Williams, Iiommes, F.A., Fed. Proc. 2l., Klingenberg, M., in B-Chance: Acad.Press, New York, 1963, Schneider, W.C., J.Biol.Chem.

(1954). J.Biol. Chem. 217, 383 (1955). Adv. Enzym. l7, 65 (1956). 142 (1962). Energy linked functions of mitochondria, pg. 121. 176, 259,(1948).

342

by

malonate.