Purification of nonbindable and membrane-bindable mitochondrial hexokinase from rat brain

Vol. 68, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PURIFICATION OF NONBINDABLE AND MEMBRANE-BINDABLE MITOCHONDRIAL HEKOKINASE FROM RAT BRAIN Philip Biochemistry

November

Received

L. Felgner Department, East Lansing,

and John E. Wilson Michigan State University Michigan 48824

24,1975

SUMMARY: MgC12-induced binding of glucose-6-P solubilized rat brain hexokinase to rat liver mitochondria has been found to be markedly diminished by increasing ionic strength. Using a modified assay of binding ability, it has now been possible to demonstrate that purified preparations of brain hexokinase do retain appreciable ability to bind to mitochondria. A slight modification of the previous DEAE-cellulose chromatography procedure (4), permits resolution of the hexokinase into two major components designated as Type Ib and Type I, based on their ability to bind and not bind, respectively, to mitochondria. Ib and I, appear to be identical in molecular size and subunit composition, but differ slightly in net charge.

It

has been demonstrated

as found

in brain,

presumably membrane

selectively

indicating which

understand

membrane

it

the

component(s)

to homogeneity the purified

chondrial a method

for

hexokinase

would

resolution

with

between

it

was not

is

the

the purpose

of purified

and to indicate

hexokinase

Although

previously

bindable

and the outer enzyme and

the enzyme has been possible

to demonstrate with

communication

why this

the mitoto describe

and nonbindable

some of the reasons

to better

the

to interact

of this

of this

In order

to purify

ability

membrane,

components)

the enzyme.

be advantageous

of hexokinase,

mitochondrial

(or

of the membrane. (4),

It

outer

component

interaction

enzyme retained

membrane.

the Type I isozyme

to the

interacts

of the

mitochondrial

that

binds

selectively

requisite

that

some specific

the nature

purified

(l-3)

forms

was not

of

observed

earlier. MATERIALS Chemicals (4,s).

and rats

Hexokinase

Copyright All rights

were

obtained

was assayed

NADP and glucose-6-P mitochondria

were

0 1976 by Academic Press, of reproduction in any form

Inc. reserved.

from

as described

dehydrogenase prepared

AND METHODS

according

commercial previously

concentrations to the method 592

sources (4)

were

given

except

doubled.

of Sottocasa

earlier that

the

Rat liver --et al.

(6).

Vol. 68, No. 2, 1976

Three

times

hexokinase

BIOCHEMICAL

washed

rat

(7) were

was purified

brain

were kinase

strength

particles

that

rat

enhanced

diluted

in place

mitochondria than

(but

by increasing

with

did

(see

liver

the

1.

not

bound

an appreciably

liver

mitochondria, ionic mitochondria),

temperature

l),

modification

to 25'.

excess

the conditions

modification:

a) hexo-

such that

exceed

0.005

mitochondria,

modifications

ionic

Fig.

(Fig.

M sucrose

These

liver

slight

of the enzyme with

the following

by increasing not with

for

hexokinase

3).

noted

0.25

solubilized Rat brain

except Fig.

medium did of rat

0'.

(4)

where

(5) with

to inhibition

particles

described.

by incubation

Except

incubation

than

activity

susceptible brain

used

rather

brain

hexokinase

were in the

were

was at 25'

sites.

previously

preparations

ionic

procedure

was assayed

binding

as described

and glucose-6-P

as previously

column

ability

mitochondrial

(5)

to Chou and Wilson

in the DEAE-cellulose Binding

particles

obtained

according

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

resulted greater

the

M, b) rat incubation

from

observations

proportion

(see Fig.

binding Binding

brain

and c)

and the binding strength

total

of the was less 1);

with

was also assays

were

done

strength

Inhibition of Binding by Increasing Ionic Strength. Binding ability of glucose-6-P solubilized hexokinase was assayed as described previously (5), using rat liver mitochondria. The indicated salt concentrations were added to the tubes before initiating binding with MgC12. Open symbols (o,A) represent KC1 and closed symbols (.,A) potassium phosphate (pH 7.0).

593

BIOCHEMICAL

Vol. 68, No. 2, 1976

in polypropylene been

dipped

microcentrifuge

in 2% bovine

boo ; coating

with

the hexokinase

AND BIOPHYSICAL

tubes

serum

(Brinkmann

albumin

BSA was found

(BSA)

to prevent

to polypropylene

RESEARCH COMMUNICATIONS

Instruments)

solution

then

artifacts

(unpublished

which dried

had

at approx.

due to adsorption

of

observation).

RESULTS From Fig.

1, it

MgC12 - induced mitochondria, assay

the first

bound

(Fig.

time

that

In this binding

When the conditions Chou and Wilson KCl,

elution

conditions,

other

at about

Fig.

2.

(4)

patterns

these

low ionic

for

are

occurs

(see Methods),

purified

hexokinase

were

severely

more than

inhibits

hexokinase used

for

at 0.02-0.03

assay

it

the

to liver the binding

M ionic

strength.

has been possible

can bind

to rat

50% of the

to

brain

enzyme was

present.

modified

such as those

g KCl,

routinely

DEAE-cellulose

slightly

two peaks 0.075

been

experiment

sites

strength

solubilized

inhibition

binding

2).

when excess

that

of glucose-6-P

percent

a modified

particles

clear

have previously

Fifty

Using show for

binding which

(5).

is

column by using

shown in

of hexokinase, have been

Fig.

chromatography a shallower 3 are

one at about

reproducibly

used by gradient

obtained. 0.065

resolved

MKCl (eight

Under and the experiments).

Binding of Pure Hexokinase. Hexokinase (0.065 units, purified according to Chou and Wilson (4)) was incubated with brain particles as described in Methods. Total volume was 0.25 ml.

594

of

BIOCHEMICAL

Vol. 68, No. 2, 1976

The relative about

3 also

at least

indicates

partly

lower

salt

refer

to these

preliminary

two forms nonbindable. analytical

in

the

the two peaks

reasons

that

bindable

concentrations

and Type In for with

of activity

75%:25% to 25%:75%; Fig.

is

amounts

AND BIOPHYSICAL

this

particles,

completely

have,

remain

the enzyme eluted

to brain is

for

while

of the

focusing

under

varied

KC1 concentrations

hexokinase

eluted reason

the bindable

eluted experiments,

from

investigation.

For this

of the enzyme as Type I b for

isoelectric

however,

at higher

nonbindable.

The asymmetry

RESEARCH COMMUNICATIONS

peaks,

at we

enzyme, together

suggest

2C

-0

10

-C

5.c

-C

25

m -C

that

VI cE 3

--

--

Frachon

Fig.

3.

number

DEAE-Cellulose Column Chromatography of Rat Brain Hexokinase. Chromatography was performed as previously described (4) except In the upper elution that shallower salt gradients were used. pattern a 600 ml linear gradient from 0.0 to 0.20 g KC1 in column buffer was used, collecting The lower elution 3.8 ml fractions. pattern was done exactly the same except the gradient went from 0.0 to 0.15 g KCl. The KC1 concentration (0) was determined by conductivity measurements on the fractions. Hexokinase activity (0) and percent bound (A) were measured as described in the Methods section. In both experiments bindable enzyme was found only in the high salt peak (0.075 g KCl). The proportion of the enzyme in the second peak which could be bound was, however, found to vary in different experiments e.g. in the lower profile, only 35% of the activity in the second peak was bindable, whereas in the experiment shown in the upper profile, 70% was bindable.

595

Vol. 68, No. 2, 1976

BIOCHEMICAL

Type I,, and Type I b are Variation

total). also

suggests

in

turn

AND BIOPHYSICAL

composed

in the percentage

that

some of these

RESEARCH COMMUNICATIONS

of more subforms

bindability

subforms

(at

least

of Type Ib (see

six

Fig.

3)

are nonbindable.

DISCUSSION It

had been

solubilization ionic

association

actually

of the it

little

enzyme with that

incorrect.

investigation)

which

for

have,

in assays

(1,

ionic

reasons

of binding

rebinding

here

(5),

Using

this

appropriate

modifications

of the binding

demonstrate

that

brain

Furthermore, cellulose

the mitochondrial

membrane,

ion

investigation.

remains

under

Type Ib)

serves

probable

that

form produced

as the

the physical

starting

point

in variable

(see Fig.

contain

observations)

amounts detectable or lipid

(5),

596

Two major

ability

so the

I

of

forms, and Ib are

n

to interact basis

for

enzyme

3) during (10,

to

DEAE-

the demonstration

with

this

distinct-

(presumably it

to some extent,

carbohydrate

used

possible

described

the purification,

at least

mitochondria

to mitochondria.

mitochondrial for

less

and making

Although

their

remain

is

has now been

or chemical

Since

which

been routinely

of the enzyme.

in

was

by liver

previously

resolved.

have

presumption

reasons

has permitted

difference

obser-

mitochondria

can bind

Type In represents,

enzyme does not unpublished

(4)

heterogeneity

by the

this

of the previously

procedure

of this

enzyme to

binding

it

of

of the

(for

is

assay,

Type In and Type Ib have been distinguished

the strength

new information,

hexokinase

modification

undetected

designated readily

slight

chromatography

a previously

than

low

would

by brain

effect

of practicality

purified

that

cause

strengths

show that

found

could

In contrast,

In view

ionic

of hexokinase

strength

8, 9).

(8).

to moderate

presented

strength

enhance

the membrane

ability.

ionic

to slightly

low

binding

to the

hexokinase

we have also

Furthermore,

susceptible

high

on the MgC12-induced

The results

mitochondria.

that

appeared

was presumed

or no effect

under

observed

of mitochondrial

strengths

vation,

previously

all

seems quite

an artifactual

purification.

The

and J. E. Wilson

difference

between

Iband

In

Vol. 68, No. 2, 1976

is

not

likely

to be due to such

purification

in that acid

(by the dansylation

by SDS-gel

principle

difference net

greater

Partial

between

charge,

with for

might

include

Ib is

a phosphorylated

dephosphorylated

(and

'

proteolysis

than

of binding

less

molecular

weight

suggest

that

affecting

charged

on its

is

In.

(enzymatically?)

negative)

seems

in N-terminal

results

and dephosphorylated is

but

in a modification

more negatively

form which thus

The latter

during

out,

to differ

or apparent

Ib and In lies

DEAE-cellulose)

RESEARCH COMMUNICATIONS

ruled

found

technique)

Ib being

phosphorylated

loss

been

electrophoresis).

affinity

resulting

factors.

Ib and In have not

(98,000

their

AND BIOPHYSICAL

of the enzyme has not been entirely

unlikely amino

BIOCHEMICAL

In during

(based

the

Such modification forms

e.g.

perhaps

converted purification

to the with

ability. ACKNOWLEDGEMENT

Financial

support

from the National

for

Institutes

this

work

was provided

by Grant

NS-09910

of Health.

REFERENCES 1.

2.

Rose, I. A., andwarms, Craven, P. A., Goldblatt,

3.

Kropp,

J. V. B. (1967) J. BioZ. Chem., 242, 1635-1645. P. J., and Basford, R. E. (1969) Bzochemistry,

8, 3525-3532. E. S.,

and Wilson,

J. E.

(1970)

Biochem. Biophys. Res. Cownun.,

38, 74-79.

4. 5. 6.

Chou, A. C. and Wilson, J. E. (1972) Arch. Biochem. Biophys., 151, 48-55. Wilson, J. E. (1973) Arch. Biochem. Biophys. 154, 332-340. Sottocasa, G. L., Kuylenstierna, B., Ernster, L., and Bergstrand, A. J. (1967) J. Cell. Biok 32, 415-438. J. E. (1973) Arch. Biochem. Biophys., 2, 543-549. 7. Wilson, 8. Wilson, J. E. (1968) J. BioZ. Chem., 243, 3640-3647. 9. Teichgraber, P., and Biesold, D. (1968) J. Neurochem. I&, 979-989. 10. Craven, P. A., and Basford, R. E. (1974) Biochim. Biophys. Acta 338, 619-631.

1

These results are consistent with earlier studies using the enzyme These prepared according to the original Chou and Wilson (4) procedure. the present observations, enzyme preparations probably were, in vimof mixture of I and I but were found to be homogeneous with regard to molecular we!?ght (en;., SDS-gels or centrifugal methods).

597