Hexokinase binding sites on mitochondrial membranes

BIOCHEMKAL

Vol. 38, No. 1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HEXOKINASE BINDING SITES ON MITOCHONDRIAL MEMBRANES*

Elin S. Kropp** and John E. Wilson Department, Michigan State University, Michigan 48823

Biochemistry

Received

October

The major

for brain hexokinase are found on the outer mitoare not present on inner mitochondrial or microsomal

portion

of the

the mitochondria using

by Katzen

et al.

remaining

activity

treatment

of the mitochondria osmotic

is not

sufficient,

indicating drial

untreated (2),

it

membranes.

hexokinase

refer

in latent

retains We have,

250-350

&,

branes

were

*Supported **Participant Summer,

after prepared

fasting

the

terminology

portion

the

is associated is readily suggested

as "overt".

b ecoming

apparent

The only

of membrane-disrupting

cycles).

therefore,

Disruption latent with

techniques

of the membranes

enzyme

in soluble

one or both

investigated

after

form

of the mitochon-

the binding

of rat

brain

membranes.

Experimental Membranes

adopting

an association

to mitochondrial

Mitochondrial

activity

(3,4),

to release

in brain

of this

to this

form

or freeze-thaw

activity

half

by a variety

however,

that

hexokinase

mitochondria;

we shall

is

shock

total

Approximately

(1).

assayable

(e.g.,

Lansing,

27, 1969

Summarv - Binding sites chondrial membrane, but membranes.

with

East

- Livers

were

overnight.

according

Procedures obtained

from Sprague-Dawley

Mitochondria

to Sottocasa

et al.

rats,

and mitochondrial (5),

using

the

by a grant from the Life Insurance Medical Research Fund. in the NSF Undergraduate Research Participation Project,

1969. 74

mem-

(4),

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 38, No. 1,197O

“3-layer”

gradient’.

in 0.25

The separated

M sucrose

for

As says - Hexokinase were

determined

Cytochrome

For

pH 7.0. sion

(6),

c oxidase

by following

(5),

were

resuspended

the decrease

c in 1.0 ml of 0.1 M potassium

phosphate

enzymes,

one unit

of substrate

one arbitrary

fluorescence

tion

(6) was suspended

bated

15 min at 25’,

defined

per min.

one unit

in 0.25

Hexokinase M sucrose

and centrifuged

the solubilized

absorbance

0.03

umole

causing

the

conver-

was assayed an increase

instrumental

- The crude

re-

- 0.5 mM EDTA,

causing

our

(7)

procedures.

in

oxidase

as that

per min under

Brain

as that

Monoamine

is defined

unit

of Solubilized

is

and protein

published

containing

(8);

containing

by previously

was assayed

fluorometrically

tant

kinase

of enzyme to a cuvette

these

of one umole

Preparation

RIDCR2 and adenylate

addition

cytochrome

membranes

use.

spectrophotometrically

at 550 q~ after duced

mitochondrial

of

conditions.

mitochondrial

frac-

- 1 mM glucose-6-phosphate,

at 41,000

X g X 15 min.

incu-

The superna-

enzyme was used immediately

for

the binding

experiments. Hexokinase were

Binding

incubated

brain

for

hexokinase

41,000

Procedure

15 min at 25’ in 0.25

X g X 15 min,

binding

in the supernatant.

expected

for

an adsorption

to the

amount

of added

a maximum with

with

M sucrose

activity

ing

- Untreated

appropriate

Hexokinase

mitochondria membranes.

After

from

directly

or membranes, In these

centrifugation

exhibited

initially

membranes

of the solubilized

the decrease

binding

being

or isolated amounts

- 3 mM MgC12.

was calculated

process,

excess

mitochondria

in hexokinase

the

behavior proportional

and asymptotically

experiments,

at

binding

approachwas

‘The 15 set sonication step was carried out with a Branson S-125 sonifier This limusing the maximum power possible (1.5-3.0 amp) without frothing. itation resulted in somewhat less extensive separation of the membrane marker enzymes than observed by some other workers (5,9) but reduced the contamination of the outer membrane fraction with inner membranes, an advantage for our purpose since we desired a reasonably accurate measure of the binding ability of the outer membrane. 2Abbreviations used: RIDCR, rotenone insensitive DPNH-cytochrome c reductase; BSA, bovine serum albumin. 75

Vol. 38, No. 1, 1970

BIOCHEMICAL

always

conducted

under

branes

or mitochondria

conditions

where

since

implied

this

Results Hexokinase membranes

to the

distributes

ties

(Table

for

the outer

activities

I)

considered

in the outer of the

chondria

with

specific excess

likely

that

branes

even if

it

effective

than

(Table tamination

of the

branes, more,

the if

accessible

sites

to be found ability fraction marker

in

penetrate

the

those

incubation

from a com-

of intact

mito-

to the outer

surprising

since

it

mem-

seems un-

sites

on the inner

tested

their

hexokinase

binding

was ten-fold

more

membrane

fraction

fraction

when compared

mem-

membrane, (the

membrane

membranes

Clearly,

significant

hexokinase

greater

binding

that

ability

total

fraction)

binding

be expected

outer

a unique

to the conclusion

76

inner

membrane from

the

of the membranes

on the is

a preponder-

of the inner

separation sites

rendered

in the

the

mem-

Further-

accessible

remaining

than would

that

binding

leading

were expect

In fact,

exceeded

suggesting

of additional

membranes

con-

outer

ten-fold.

sum of the newly

fraction

in exposure

than

one might

fraction.

somewhat

appreciable

the avidly-binding

be much greater

outer

on a per mg basis

indicated

with

on the outer

membrane,

for

on the inner

ability

enzyme distribution,

mitochondrial

apparent

to binding

fraction

sites

results

outer

outer

must

membrane

and was actually

activi-

of these

enzyme distribution

of the

the inner

of the outer

were

membrane

binding

plus

oxidase

Concentration

in binding

not

of the

markers

readily

results

of course,

membrane

binding

to separation

present.

that

by removal

ance of the total membrane

hexokinase

difference

additional

enzyme.

as characteristic 5,9).

Thus

the marker

actual

(e.g.,

values.

membranes

inner

of bindable

to the RIDCR and monoamine

activity

is,

to added mem-

prior

is

the inner

Since

I).

mitochondria

fraction

they were

was found

an excess

membrane

enzyme could

When the separated ability,

intact

membrane

This

the

was proportional

by many investigators

brain

exclusively.

it

and Discussion

identically

mitochondrial

parison

brane

bound

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

membrane. property both

of the the

“overt”

three

The mitochondria inner and outer

were incubated membranes.

with

'The total hexokinase binding capacity tions; hexokinase binding is expressed protein.

b

given

---

+ 0.20

em-

f 0.02

i 0.07

;t 0.08

hexokinase

are

0.41

0.25

0.17

0.16

prior

with

(

---

I

I

0.41

0.50

1.00

0.49

0.36

of the

mm-

f 0.16

+ 0.07

f 0.15

f 0.16

f 0.01

of the membrane fracbound per mg membrane

to separation

)

Outer Membrane

deviations.

2 0.36

i 0.02

r 0.04

f 0.03

standard

0.56

0.05

0.12

0.07

0.05

2 0.02

Inner Membrane

Activities

and Protein

units mg protein

Sites,

Fractionsa

Binding

was determined after isolation as units of hexokinase activity

brain

experiments

13 f 8

12 i 9

55 f 10

45 f 15

48 f 11

48f3

Specific

Membrane

Hexokinase

Intact Mitochondria

Mitochondrial

Activities,

Outer Membrane

excess

to seven

87 f 8

Protein

aThe average

88 f 9

Cyt c Oxidase

from

Binding

Hexokinase

values

55 + 15

Oxidase

Monoamine 45 + 10

52 2 11

Inner Membrane

RIDCR

Si.tesC

and Outer

$ in Fraction

Inner

of Enzymatic

52 f 3

I

Between

Distribution

Bound HexokinaseD

TABLE I.

s 0

.

8.-E 5f

Vol. 38, No. 1, 1970

and “latent”

BIOCHEMICAL

forms

of brain

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hexokinase

mitochondrial

membrane.

Craven

(9) who f ound the hexokinase

et al.

outer

membrane

fraction

Our previous required

able

prepared

in 1% BSA caused

change

in

the

from disruption

1% BSA is

essentially

Osmotic exposure kinase, (5).

either latent

a marker

for

both

could

becoming

exposed

of the only

membrane

The current

controversy

to revolve

the

ambiguity

somes.

both

outer

results),

somal

membranes. which

the

by the

pressure

expect

of

osmotic membranes

results

in Table

II.

or in 1% BSA, resulted and the release

in order

membrane,

to the exterior

of the extensive

space the

binding

latent

sites

or that is

in

of adenylate

to expose

the enzyme occupies

the

on

activity

somehow masked,

disorganization

absence

the use of monoamine

mitochondrial resulting

the outer

Rose and Warms (11)

lished

erty

in

expected

of the intramitochondrial

surrounding

the

activities

detect-

likely

disruption.

for

these

without

indeed

been

of the mito-

the osmotic

water

of the outer

as the result

the

hexokinase

the mitochondrial

demonstrated

enzyme bound

RIDCR as markers about

That

disruption that

and the outside

portion

to accompany

either

activity

Since

as expected,

membrane

indicate

the inside

of a major

for

in

has recently

suspension

one would

portion

of

of the particles

in pure

hexokinase, the soluble

A requirement

activity

is

report

brain

(4),

that

water,

by suspension

of the

of the latent

properties

procedure

recent

the outer

mitochondria.

of the latent

of pure

with

concentrated

membranes

to have occurred.

by this

shock,

brain

of the membranes. that

the

activity

(9) wh o reported

scattering

of the particles

-are disrupted

with

exposure

exposure

light

to result

lysis

from

that

--et al.

be associated

consistent

of the mitochondrial

by Craven

chondria

is

suggestion

disruption

questioned

This

must

from the

mitochondrial

have reported, of significant

Thus hexokinase

may be used unequivocally

membrane

binding

apparent membrane

5,9,lC) location

hexokinase ability

binding

appears for

and seems of

and the micro-

and we have confirmed

as a marker

‘78

(e.g.,

oxidase

(unpubby micro-

to be one prop-

the outer

mitochondrial

Vol. 38, No. 1, 1970

TABLE II.

BIOCHEMICAL

Effect

of Osmotic

Kinase

on the Hexokinase

of Brain

Medium

:ok :inase (units) I Particulate hrert

Latent

and Adenylate

Mitochondriaa

Hex

Soluble

Suspending

Shock

Activities

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Adenylate

Total

Solubl

Kinase

(units)

Particulate

Total

0.25 M Sucrose

0.21

1.09

0.82

2.12

1.16

6.64

7.80

Water

0.25

2.26

0.10

2.61

6.12

2.54

8.66

l$ BSA

0.10

2.44

co.05

2.54

6.28

2.86

9.14

0.5% Triton in 0.23 M Sucrose

0.82

1.35 co.05

2.17

7.44

0.88

8.32

aThe crude mitochondrial fraction was prepared as described in the text, and portions suspended in the indicated media. After centrifugation at 41,000 X g X 15 min, the supernatants were assayed for "soluble activities". The pellets were resuspended in 0.25 M sucrose and assayed for "overt" hexokinase. After treatment with 0.s Triton X-100, the particulate fractions were again assayed for hexokinase and for adenylate kinase. The increase in hexokinase activity after addition of Triton is defined as "latent" activity.

membrane. tact

The technique

mitochondria

described

above,

before

of binding preparation

seems particularly

hexokinase

to the outer

of the submitochondrial

membrane

of in-

fractions,

as

convenient.

References 1. 2. 2:

5. 6. 7.

8. 9. 10. 11.

Johnson, M. K., Biochem. J., '& 610 (1960). Katzen, Ii. M., Soderman, D., and Wiley, C., Fed. Proc., 28, 467 (1969). Teichgraber, P., and Biesold, D., J. Neurochem., Q, 97971968). Wilson, J. E., Biochem. Biophys. Res. Commun., 28, 123 (1967). SOttocasa, G. L., Kuylenstierna, B., Ernster, L., and Bergstrand, A., in Methods in Enzymology S. P. Colowick and N. 0. Kaplan (Editors), Vol. X, Academic Press, New York, 1967, p. 448. Wilson, J. E., 3. Biol. Chem., &, 3640 (1968). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem., E, 265 (1951). Tipton, K. F., Anal. Biochem., 28, 318 (1969). Craven, P. A., Goldblatt, P. J., and Basford, R. E., Biochemistry, 8,

3525 0969).

D. W., Harris, R. A., and Tan, W. C., Biochem. Green, D. E., Allmann, Bionhys. Res. Commun., 2, 368 (1968). Rose, I. A., and Warms, J.V.B., J. Biol. Chem., 242, 1635 (1967).

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