Brain mitochondrial hexokinase: Solubilization by adenine nucleotides and in coupled respiration

Vol. 54, No. 4, 1973

BIOCHEMICAL

AND BJOPHYSICAL RESEARCH COMMUNICATIONS

BRAIN MITOCHONDRIAL HEXOKINASE: SOLUBILIZATION BY ADENINE NUCLEOTIDES AND IN COUPLED RESPIRATION M. Seth

Hochman and Bertram

Sacktor

Laboratory of Molecular Aging, Gerontology Research Center, National Institute Child Health and Human Development, National Institutes of Health, Baltimore Hospitals, Baltimore, Maryland 21224

Received

September

5,

of City

1973

SUMMARY: The relative potencies of ATP and ADP in debinding rat brain mitochondria hexokinase were measured. At fixed total concentrations of adenine nucleotides, added exogenously, solubilization of the enzyme increased as the proportions of ATP to ADP were raised. The generation of physiological concentrations of ATP by the mitochondria during coupled respiration resulted in a 2-fold increase in solubilization. These findings support the hypothesis that the cytosol-mitochondrial compartmentation of hexokinase may be a factor in the regulation of hexokinase activity and glycolysis. INTRODUCTION: controlling mostly

Earlier the cerebral

associated

with

reversible

binding

ATP and,

to a lesser

posed

studies

(6)

that

have indicated

glycolytic

rate

mitochondria

control

distribution.

stems

that

is

observations

several-fold

lower

of the particulate

for

that

cytosolic coupled

in (5-7);

however, that

glucose.

However, is able

the association

This

question

that

the mitochondrial

the

hexokinase

hexokinase

would

(9);

and

the ATP generated enzyme

remains

to the physiological

be exposed

oxidative

to phosphorylate

the mitochondrial

relevant

the response

Km for ATP relative

during

of the enzyme to the mitochondria

may be particularly

hexokinase

see (8,lO).

as to whether

to solubilize

hypothesis

is slower

the ATP generated

the question

has been pro-

(7,8);

to have a lower

undergoes

glucose-6-P,

soluble

enzyme

is

by regulating

of this

glucose-6-P

cases

It

be accomplished

the mitochondrial

some

with

in

activity

hexokinase

(5,6).

for

can be used by the mitochondrial

respiration

regulate

may

to the inhibitor

enzyme

hexokinase

The attractiveness

Rose and Warms (5) have shown phosphorylation

activity

for

enzyme appears

the soluble

the enzyme

the Ki of glucose-6-P

hexokinase

the mitochondrial to that

than

In brain,

has a key role

of mitochondria

ADP solubilize

of hexokinase

hexokinase

The particulate

incubation

the soluble-particulate from

(1,2).

(3,4).

and debinding; extent,

that

to varying

and,

during thus,

to be resolved. situation,

concentrations

in of

Vol. 54, No. 4, 1973

BIOCHEMICAL

ATP and ADP, depending cation

we present

mitochondrial

METHODS: methods

evidence

brain

(13).

5 mM Tris-HCl,

intact

and other

membrane

published

rapid

of 6 and 2.5,

fragments

of figures x g for

bilized

were

and table.

Reactions

hexokinase,

contained

2.1 umoles

of NaF, 4.1 umoles

Aliquots

according

the

in detail

75 mM sucrose,

serum albumin,

the mitochondria

were

by myelin,

respiratory obtained

adjusted ultra-

synaptosomes

favorably

with

control with

of ATP,

other

ratios

glutamate

and

was measured

were

at least

for

of hexokinase

defined

8% perchloric

acid.

previously

2.1 I&I glucose,

with

Assay

mixtures

of 0.62

1547

pmoles

The rate

Initial

the

of

rates enzyme.

formation

of

conditions. were

in the KOH-neutralized

4.6 mM NADP, 1.4 units

dehydrogenase.

ml.

to mitochondrial

the reactions

contained

20.7

of NADP, and 0.7 unit

catalyzing

these

was

mixtures

pH 7.4,

at 340 nm.

as the activity

ATP was measured

(15).

volume

the solu-

Reaction

4.6 pmoles

at

Hexokinase

buffer,

proportional

ATP was estimated,

4.1 RIM MgC12,

of glucose-6-phosphate

in a final

containing

(14).

of Tris-HCl

of MgC12,

per min,

in which

by centrifugation

assayed.

and Crane

5 min and directly is

were

spectrophotometrically

of glucose-6-phosphate

In experiments

pellets

25 pmoles

dehydrogenase

terminated

in approprfate

of the supernatants,

4.1 pmoles

NADPH formation

out as detailed were

to Hernandez

of glucose,

of glucose-6-phosphate

linear

carried

and the resuspended

at 30",

described

bovine

of 2.7 and 1.5 were

added

as described

225 mM mannitol,

Typically,

incubations

5 min at 0".

determined

with

ratio,

and modifying

low and compared

procedures.

and ADP:O ratios

(12),

of the preparation

was relatively

brain

respiration.

by combining

(13),

communi-

respectively.

legends

1 pmole

mitochondrial

crystalline

microscopy

and contamination

preparative

Mitochondrial

One unit

in the ATP:ADP

medium contained

As seen by electron

structurally

of rat

and Jobsis

0.2 mM EDTA, and 4 mg/ml

to pH 1.4.

in the compartmentation

prepared

and Moore

The isolation

In this

of changes

were

RESEARCH COMMUNICATIONS

of the cell.

of coupled

mitochondria (11)

activity

a change

as a result

of Ozawa -et -*al

succinate,

for

and as a consequence Rat

elsewhere

40,000

on the metabolic

hexokinase

exogenously,

AND BIOPHYSICAL

25 mM Tris-HCl, of hexokinase

terminated extracts,

as

pH 7.4, and 0.7 unit

Vol. 54, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I EFFECT OF ATP AND ADP ON THE SOLUBILIZATION OF RAT BRAIN MITOCHONDRIAL HEXOKINASE EFFECTOR

PARTICULATE

SOLUBLE

TOTAL

mUnits ATP

50

37

87

ADP

82

5

a7

Mitochondria (89 munits, 0.46 mg protein), suspended in 0.5 ml of 225 mM mannitol, 75 mM sucrose, 0.2 mM EDTA, 5 mM Tris-HCl, and 4 mg/ml crystalline bovine serum albumin, adjusted to pH 7.4, were incubated for 5 min at 30" with 12 mM glutamate, 3.3 pg oligomycin and 10 mM ATP or ADP in a final volume of 0.63 ml. Total hexokinase activity is the sum of the measured particulate and solubilized activities, determined as described in the text. The hexokinase activity solubilized in the absence of added nucleotides was subtracted from the soluble values.

Protein

was determined

crystalline

bovine

serum albumin

were

of rat

tested

is noted

brain

more hexokinase

experiments.

The ATP-solubilized

originally

the

"latent"

not

evident.

however. suspensions described experiments

are

The discrepancy

activity

as the locus

effectiveness range

when both

nucleotides

period

with

with

and shorter

in these

of Purich

(19),

incubation

concentrations 1548

the

(17),

was

and Fromm (18),

from our mitochondrial (13),

recently

or the use in

the present

conditions.

of ATP to ADP as debinding

of nucleotide

total

experiments

by Wilson

synaptosomes

hexokinase

solubilization,

approximated

Thus,

those

It

by ADP in different

activities

as described

in agreement

of 5 min.

from 30 to 50% of the

incubation.

mitochondria,

of the "latent"

of more physiological

an appreciable

comprised

the

of ATP and ADP

by ATP than

and soluble

contamination

with

potencies

at promoting

may he due to the absence

of significant

(16),

an incubation

solubilized

into

in brain

Our results

The relative over

being

introduced

activity

hexokinase,

more effective

The sum of the particulate

activity.

the relative

of 10 mM with

ATP was considerably

--et al.

standard.

I compares

at a concentration

that

of Gornall

mitochondrial

4- to 7-times

activity

as the

Table

RESULTS AND DISCUSSION: as solubilizers

by the method

agents (Fig,

1).

was maintained Control

experi-

BIOCHEMICAL

Vol. 54, No. 4,1973

OV--

:

0

o-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 10

5 CONCENTRATION

oi:,-

I

(mM)

I 10 INCUBATION

20 TIME

30 (mln )

Fig. 1. The debinding of cerebral mitochondrial hexokinase at different concentrations of ATP and ADP. Mitochondria (50 munits, 0.61 mg protein) in Table I, were incususpended in 0.5 ml of isolation medium, described bated for 5 min at 30" with 12 mM glutamate, 41 pg oligomycin, and the indicated concentration of adenine nucleotide in a final volume of 0.62 ml. The hexokinase activity solubilized in the absence of added nucleotide (7 munits) was subtracted from each plotted value. Fig. 2. The time course of solubilization of brain mitochondrial hexokinase. Mitochondria (27 munits, approximately 0.3 mg protein) suspended in 0.25 ml of isolation medium, described in Table I, were incubated at 30" for the indicated times with 12 mM glutamate, 40 dl potassium phosphate, 21 pg oligomycin, and 9.7 mM ATP or ADP in a final volume of 0.31 ml. The hexokinase activity solubilized in the absence of added nucleotide was subtracted from each plotted value, e.g. at 30 min, 4.6 munits were solubilized in the control incubation.

ments ruled

out

supernatants

the possibility

after

of sufficient

incubations

of hexokinase

Solubilization

which

with

when most

Since

and ADP might ratios

vary

of ATP:ADP,

of the mitochondrial

ADP were

of the supernatants

found

due to carry

to inhibit

of incubation

In the experiment

(Fig.

2).

approximately

in the over

the subsequent

Solubilization

cell

the

constant

according at a fixed

by ATP was slowed

hexokinase

hexokinase

total

the relative

to the metabolic nucleotide

was determined.

1549

nucleotide

illustrated,

in

enzyme was

at the

longer

incubation

had been solubilized.

concentration

whereas

total

of adenine

80% of the mitochondrial

of the mitochondrial

essentially

with

activities

concentration

in the intact

remain

hexokinase

of the enzyme at a fixed

in 30 min.

periods,

lower

(21,22).

9.7 mM ATP was used,

solubilized

would

time

the

of mitochondria

ADP in the aliquots

measurements

increased

that

state,

of adenine concentrations the effect

concentration, Pig.

nucleotide of ATP

of different on the solubilization

3 describes

an experiment

Vol. 54, No. 4, 1973

in

which

the

increased

total

nucleotide

linearly

illustrated, 2.8

BIOCHEMICAL

with

were

mM.

Of

obtained

of

In

experiments,

present

ATP

and

the

solubilization

of

0.8

mM ATP:4.8

mM ADP.

that

tionally

the

of

sum

that

the the

and

4A

would

solubilized

was

increase

respiration,

not

that

increase

in

0.75

mM,

0.8

mM ADP

as

did

the

reciprocal

data

in

a combination

by

each

Figs.

3 and of

ATP

nucleotide,

however,

experiments

were

the

not

or

respectively. promoted

4-times

ratio

of

1 reveals and

ADP

tested

that

5.6

intracellular

and

by

ATP

the

was

addiapproximated

separately

at

concentrations

saturating

of

[see,

ATP

attained

to

be no

adenine

Fig.

1

a decrease

increase

in

concentration

studies

(6,18)

itself,

nor

debinding

increase

a concentration

nucleotides

than

0.8 in

did

rat

brain,

the

formed

of mM.

ATP

of

in

These --in

the

was

enzyme the

hexokinase

mM and

vivo

1550

the are

(18).

Pi Pi

coupled

no

signifiof

solubilization the

data

correlated incubation.

in

Fig.

with

an

Perhaps was

relatively

was the

4B,

of

found

concentration

approximately The

again,

during had

activity ADP

soluble

compartmentation

by was

in

hexokinase

enhanced

evident

during

values

soluble

that the

only

activity;

of

alter

soluble 4.7

in

hexokinase

differing

increase

concentration

it

the

particulate

demonstrated

of

ATP

in

That

level

of

The

increase

in

coupled

incubation,

oligomycin.

The

during

incubations,

decrease

seen.

of

parallel

nucleotides.

elevated

more

in

the

mitochondria

15 min

by

by

of

by

Z-fold

found

was

result

by

After

inhibited

activity

an

this

generated

that

matched

by

the

ATP

hexokinase.

previous

with

significance,

adenine

and

the

not at

mM ATP

the

noted,

these

double

the

induced

showing

of

be

that

action

associated

mated

in

total

since

hexokinase

when

used

precisely

solubilizing

was

should

of

in was

cant

It

almost

was

activity

of

released

release

synthesis

hexokinase no

4.8

results,

maintained

that

4.4

hexokinase

A comparison

demonstrates

respiration

that

of

was

were

solubilization

Similar

estimated

brain

shown,

(6)].

Fig.

in

rat

solubilized

nucleotides

Wilson

in

(18)

As

ATP.

nucleotide

Fromm

mitochondrial

activities

10 mu. of

total

a ratio

hexokinase

concentration.

adenine

the

and ADP

was

proportions

when

Purich

concentrations the

concentration

increasing

interest,

AND BIOPHYSICAL RESEARCH COMMUNlCATiONS

esti-

concentrations small

amount

BIOCHEMICAL

Vol. 54, No. 4, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

oi---i-

L-I 25

50

75

100

96 ATP

Fig. ratios bation

of

The solubilization of ATP and ADP, conditions were

3.

ATP

found

presence

of

associated

in

lization

of

the the

experiments, have

would

be

too

by

continuous

the

produce

was

soluble-mitochondrial

glucose-6-P

the

soluble

of in

enzyme

inactive.

Thus, and other

(21) when

when

ATP

to

the

phosphorylation

the

are

only

This the

tend

to

1 ti

earlier ATP

concentration

present

experiments was

prevented

the

hypothesis

support

hexokinase

may

of

ATP.

least

the

Ki

lo-times

be

soluble of

soluble

The of

debinding

of

enzyme

and,

hence,

glycolysis

low,

the

the

was

found

inhibitor he

would of

in

concentration

this

may

rebinding

a factor

enzyme

enzyme

1551

the

mixture.

concentrations

the

solubiin

anaerobiosis

of

high,

glucose

of

In

A doubling

at

respiration

demonstrate

a maximum

communication

are

levels

to

mitochondrial

However,

incubation,

that

levels

of

anaerobiosis.

reaction

, suggesting

of

due

coupled

unable

and of

the

this

by

debinding.

physiological

phosphorylation hand,

onset

the

is

was

substrate

activity.

ATP

to

phosphorylation.

during

(2,18)

or

ratio

who

distribution

brain

probably

compartmentalization

ATP:ADP

measurable

in

(2.2)

the

(6),

as the

hexokinase

generation

the

of

reported

of

in

oxidative

produced

the

of

the

to

was

by different 10 mM. Incu-

of

a-ketoglutarate.

used

before

findings

the

On

was

4B)

(Fig.

mitochondria

Wilson

oxygenation

regulation

favored

of during

The

with

the

a change

formed

small

5 mM ATP

in

altering

report

enzyme

been

almost the

of

succinate

could

oligomycin

of of

a result

with

of

oxidation

finding

as

contrasts

kinase

the

present

hexokinase

presence

adenylate with

The

that

the

of brain mitochondrial hexokinase a total nucleotide concentration described in Fig. 1.

at as

hexokinase

for

vfrtually would be

be slowed, by Mg

2+

Vol. 54, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TIME

(mln)

Fig. 4. Solubilization of rat brain mitochondrial hexokinase by ATP generated Mitochondria (102 munits, 0.61 m g protein) during oxidative phosphorylation. suspended in 0.85 ml of isolation medium, as described in Table I, were incubated at 30" with 22 mM glutamate, 24 mM potassium phosphate, 5.5 mM ADP, and ?: 3.3 pg oligomycin in a final volume of 0.98 ml. The reaction mixtures were flushed with 100% 02 throughout the incubation. A. Hexokinase activities. of the ATP produced during the incubations in parallel experiments B. Measurement

-

~~~~~ (5,6)

would

be favored,

a mode is

especially

neau

that

(23)

The rebinding lead

hexokinase

in normal

brain

be envisioned

glycolytic

enzymes.

found

in other

In all

these

the possible

role

additional

hexokinase

cerebral tend

mitochondrial-bound heart,

intestinal

of alterations flux

of glycolytic

assessment.

1552

should

and Passon-

activity

A coordinated

of ATP would

to brain, retina,

for

Such

in an inhibited

phosphorylation.

levels

including

in the regulation

total

inhibition. of Lowry

predominantly

postulated

high

In addition

tissues, tissues,

in that

is

of the

in glucose

and phosphofructokinase,

from glucose-6-P

of the calculation

percentage

increase

may also

require

in view

of even a small

of hexokinase

in relief

attractive

to a significant

hexokinase

resulting

metabolism to inhibit

could control (1,2), both

hexokinase mucosa,

state.

is

and liver.

in binding-debinding be considered

and will

of

Vol. 54, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Lowry, 0. H., Passonneau, J. V., Hasselberger, F. X., and Schulz, D. W.. J. Biol. Chem. 239, 18-30 (1964). Sacktor, B., Wilson, I. E., and Tiekert, C. G., J. Biol. Chem. 241, 50715075 (1966). Crane, R. K., and Sols, A., J. Biol. Chem. 203, 273-292 (1953). Johnson, M. K.. Biochem. J. 77, 610-618 (1960). Rose, I. A., and Warms, J. V, B., J. Biol. Chem. 242, 1635-1645 (1967). Wilson, J. E., J. Biol. Chem. 243, 3640-3647 (1968). Karpatkin, S., J. Biol. Chem. 242, 3525-3530 (1967). Tuttle, 3. P., and Wilson, J. E., Biochim. Biophys. Acta 212, 185-188 (1970). Kosow, D. P., and Rose, I. A., Biochem. Biophys. Res. Comm. 48, 376-383 (1972) Gots, R. E., Gorin, F. A., and Bessman. S. P., Biochem. Biophys. Res. Comm. 49, X49-1255 (1972). Ozawa, K., Seta, K., Takeda, H., Ando, K., Harida, H., and Araki, C., J. Biochem (Tokyo) 59, 501-510 (1966). Moore, C. L., and JobsIs, F. F., Arch. Biochem. Biophys. 138, 295-305 (1970). Hochman, M. S., Shimada, Y., and Sacktor B., submitted for publication. Hernandez, A., and Crane, R. K., Arch. Biochem. Biophys. 113, 223-229 (1966). Sacktor, B., and Hurlburt, E. C., J. Biol. Chem. 241, 632-634 (1966). Gornall, A. G., Barawill, C. J., and David, M. M., J. Biol. Chem. 177, 751766 (1949). Wilson, J. E., Biochem. Biophys. Res. Comm. 2, 123-128 (1967). Purich, D. L., and Fromm, H. J. J. Biol. Chem. 246, 3456-3463 (1971). Wilson, J. E., Arch. Biochem. Biophys. m, 96-104 (1972). Sols, A., and Crane, R. K., J. Biol. Chem. 206, 925-936 (1954). Fromm, H. J., and Zewe, V., J. Biol. Chem. 237, 1661-1667 (1962). Criss, W. E., Arch. Biochem. Biophys. 144, 138-142 (1971). Lowry, 0. H., and Passonneau, 3. V., J. Biol. Chem. 23q, 31-42 (1964).

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