Affinity modification of creatine kinase and ATP-ADP translocase in heart mitochondria: Determination of their molar stoichiometry

Vol.

134,

January

No. 14,

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1986

Pages

359-366

AFFINITY MODIFICATION OF CREATINE KINASE ATP-ADP TRANSLOCASE IN HEART MITOCHONDRIA: DETERMINATION OF THEIR MOLAR STOICHIOMETRY

AND

A.Y.

and

Bioenergetics, of 3 Cherepkovskaya

Laboratory Cardiology, Received

Kuznetsov

November

29,

Y.A.

Saks

USSR Street

15a

Research , Moscow

Center 121552,

for USSR

1985

dialdehyde analogs of ADP or ATP (oADP and Oxidized oATP) were shown to inhibit irreversibly adenine nucleotide translocator (T) and creatine kinase (CK) in heart mitochondria. with Inactivation of T and CK was parallel carboxyatractyloside - sensigive and (ADP + phosphocreatine) - sensitive incorpo ation of o[ H]ADP into mitochondria, respectively. o[ 5 H]ADP incorporation sensitive to CAT or ADP+phosphocreatine was used to determine T and CK contents in ,mitochondria. T content in from rat, cardiac mitochondria rabbit, and chicken was dog, calculated to be 2.6 - 2.9 moles/mole cyt.aa The same value of Tlcyt .aa3 ratio was found in liver mitoc 2’ ondria with lower cytochrome aa content. In all types of cardiac mitochondria CK content was fzund to be 2.4 - 2.6 moles/mole cyt.aa . The data show that T and CK are present in molar ratio 1:l in3all types of 0 1986 Academic Press, Inc. cardiac mitochondria.

ATP-ADP coupled

translocase

in

heart

mitochondria

phosphocreatine

proteins

to in

there

are

molar

ratio very

nucleotide

such

reagent number

of

using

as In

(4).

oADP

are

content

the

irreversible

In

of the

have ATP

present inhibitors

labelled

of

is an

a

rare

increasing

investigated

ADP

work

adenine

with

been or

kinase

of

hand,

both

However,

which other

is

creatine

determined

enzymes

the

of

(3).

a II d

the

of

presence

of

on

derivatives (5-6).

oATP

the

determination

usually

years,

cycle interaction

[ 35S]carboxyatractyloside

binding

2,3-dialdehyde

and

is

recent

nucleotide

correspondingly)

on

functionally

initial

mitochondria

mitochondria,

translocase

inhibitors

heart

are

effective

supposes

in

data

the

Their

one

I:1

few

heart

in

if

kinase

form

(1,2).

explain

only

content

and

shuttle

easy

most

creatine

and

(oATP

we have creatine

by

and

oATP,

shown

that

kinase 0006-291X/86

359

and $1.50

Copyrighl 0 1986 by Academs, Press, Inc,. AN rights of reproducfmn in any form resenwi,

Vol.

134,

No.

ATP-ADP used cardiac

to

BIOCHEMICAL

1, 1986

translocase

,

determine

molar

and

these content

AND

BIOPHYSICAL

compounds of

the

RESEARCH

have proteins

been

in

COMMUNICATIONS

successfully question

in

mitochondria.

MATERIALS

AND METHODS

Mitochondria were isolated from the heart muscle as described earlier ( 2 ) . Liver mitochondria were isolated according to ref. were obtained by a method of ( 1 1. Mitoplasts Schnaitman and Greenwal t of the inner ( 8 1. The intactness mitochondrial membrane was tested by inhibition of the respiratory rate in state 3 more than 85% by carboxyatractyloside 50 pM. Creatine kinase activity was determined as (CAT) indicated earlier ( 2 ). Oxygen consumption rate was determined polarographically using a Yellow Spring Clarck electrode ( 2 ). Cytochrome aa content was determined spectrophotometrically at wavelen th r 605-630 nm using an extinction coefficient value 24 mM -’ cmpBs ( 9 ) oATP and oADP synthesized by a per iodate method (10 ) and purified on Ieigphadex G-10 column. The final contentfation of oATP and oADP was determined at 258 E=14.9 mM cm nm, ( 11 I. Activity of ATP-ADP translocase was determined by a method described by Chan and Barbour ( 12 ). Inactivation of ATP-ADP translocase in the presence of 5-200gM oADP or oATP was assessed by determination of changes in the state 3 consumption rate or directly by changes in the rate of [ %yKF uptake. Inactivation of creatine kinase by oATP or oADP was determining detected by changes in the enzyme activity after incubation of mitochondria (lmg/ml) in a medium containing 20 mM HEPES-Na, pH 5.5-7.5 , 20 mM Tris-HCl, pH 7.5-9.0 , 3 mM Mg acetate, 0.05-1.5 mM inhibitor. After definite time intervals of incubation (O-60 min) samples were withdrawn, diluted 100-200 times by activity assay medium and enzyme activity was determined. Binding of o[~H]ADP to translocase and creatine kinase in heart mitochondria was determined in a medium containing 20 mM HEPES-Na, pH 7.0, 3 mM Mg acetate, 10 mkg/ml oligomycin, 0.3 mg incubation (1.2 mg/ml) mitochondria and 0.7 mM o[~H]ADP. After the mixture was filtered through Millipore filters for O-40 min. (0.45 mkm), filters were washed three times by a cold incubation dried and dissolved in 10 ml of dioxane scintillator for buffer, counting in “LKB Rack Beta 1 15” counter. For radioactivity specific binding of o[ ?3H]ADP with adenine determination of translocase the experiments were repeated in presence nucleotide in radioactivity was taken to of 50 PM CAT and th 8 difference show the amount of o[ H]ADP bound to ATP-ADP translocase (see the “Results” section). Similarly , to determine the specific binding in total with creatine kinase in mitochondria a difference o[~H]ADP binding and that in the presence f 0.5 mM MgADP and 15 mM phosphocreatine was determined. o[ s H]ADP binding with kinase determined after isolated and purified creatine was incubation of enzyme (0.5 mg/ml) with 1 mM of reagent for 35 min gelat 30’ C and after removal of an excess of the reagent by filtration on Sephadex-G25 fine . Removal of creatine kinase from the membrane of mitoplasts was performed by incubation of mitoplasts (5 mg/ml) for 30 min in a medium containing 0.125 M KCl, 20 mM HEPES-Na, pH 7.4 , 0.3 mM 20 mM ADP, 4’ C. dithiothreitol, 1 mg/ml bovine serum albumin , The mixture after incubation was diluted by solution containing 360

Vol.

134,

No.

BIOCHEMICAL

1, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

10 mM Tris-HCl, pH 7.4, 0.2 mM EDTA, 1 mg/ml M sucrose, and centrifuged for 10 min at 10 000 g. The bovine serum albumin procedure was repeated twice and the pellet obtained was resuspended to a concentration of 50-80 mg/ml. Creatine kinase was isolated and purified from rat heart mitochondria according to a procedure discribed by ( 13 ). Materials Enzymes used in the coupling enzyme assays, -_-------. nucleotides, creatine, phosphocreatine, HEPES,bovine serum albumine, sucrose, dithiothreitol, malate, EGTA, trypsin, trypsin were purchased from “Sigma”,USA. Digit nin, EDTA, inhibitor, glutamate were from “Serva” , FRG and [ 8 H]ADP from Tris, “Amersham” , England. 0.3

RESULTS Incubation ADP

or

ATP

nucleotide

rat

heart

the

uptake

of

mitochondria

resulted

in

and

mitochondria

with

of

[ 3H]ADP

inhibition

could

be

respiration

(Fig.1)

.

dependence

of

state

The

character

interaction

of

with

rapid

translocase

the

AND DISCUSSION

less

shows

than

in

dependences and

double

that

10 min

adenine

of

at

30’

state

3

plots

the

concentration.

a competitive be

C This

of

on ADP

may

of

control.

reciprocal

rate

of

incubation

inhibition

shows

oADP

for 1%

from

both

After

oADP

3 respiration

ADP

between

of

seen

Fig.1

of

kinase.

2OOpW

was

derivates

inactivation

creatine

easily

t hese

2,3-dialdehyde

type

taken

to

of

indicate

0.275

0.165

0.065 0

-1 l/tROPl,mn

Fig.1.

The

double

reciprocal

of different (indicated respiration

the at

rate

uncoupled

state

in in

presentation of the 3 respiration upon ADP oADP concentration in mM at straight lines). the presence of ADP was

respiration.

361

dependence

of

concentration

the The corrected

rate

medium of for

Vol.

134,

No.

binding

of

oADP

nucleotide .

Fig.

1

were

was

parameter

nucleotide When

BIOPHYSICAL

binding app. Km values for

inhibitor

obtained

giving

for

was

oATP

RESEARCH

sites

in

adenine

obtained

from

concentration,

= 0.065

(oADP)

found

of

ADP

(oADP)

Ki

COMMUNICATIONS

similar

mM.

a The

value

experiments

to

be

mM.

inactivation

at

Pig.2.

The of

the

activity

first

order

was

of

the

different

mitochondrial oADP

the

time

of

the

reaction.

The

depend

to

enzyme’

a

on

inhibitor

enzyme

(E inact.

rate

with

pK - 6.9 of

creatine

kinase

E’I

and

phosphocreatine

the

enzyme,

Similar

be

most

this

‘MgADP

be

involved

purified

radioactive

label

to

suppose

the ( %Yz)

value

of

noncovalent of

),

an

with these

a data

complex

inactive

mM)

against was

which the

formation

( 15 of

enzyme into

the

was

was

form

of

that

some

leads shows

creates

of the

transition

to

also

inactivation

a mixture

that its

by

MgADP

(0.5

“working”

mM)

state

state

of

complex

). inactivation

parallel

with and

362

on

protonation

creatine

protein,

dependent

revealed

Fig.2

protected

aitochondrial the

whose

inactivation.

characteristics

of

us

( 14

dependence

effective (15

Inactivation

of

inactivation

of

including

E’creatine’POS

logarithm

Finact

kinase

enzyme

could

-2

the

to

formation

4

may

the

and

substrates,

soluble,

G

study

acceleration

of

l/[oADP]

formation

before

creatine

of

Quantitative

groups

of

the

1:

.E + I The

the

in

on

half-inactivation

According

(E.1)

-

of

shown

dependent

allows

time

linearly

probability

is

dependence

reaction

ordinate-intercept.

point

concentrations wzis

linear

kinase

creatine

inactivation

oADP;

the

to

positive

heart

of

on

found

of

rate

concentration

PH.

AND

against

Time-course

the

the

plotted

line

this

0.14

to

translocase.

straight of

BIOCHEMICAL

1, 1986

complete

were kinase

seen was

incorporation inactivation

when used. of

the was

Vol.

134.

No.

8lOCHEMlCAL

1, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

100 so 60 70 60 50 fI 40 t; : 30 : 20

1.36 ’ 0.92 10

0

pig.2.

when

dimeric

2

enzyme Figure

with

adenine

shows

minutes

kinase

were

completely

cyt.aa

of

mitochondria.

50

incorporation

(Fig.3).

binding

to

difference, B cat

in

the

translocase cyt

.aa 3’

consistent

Since

This with

of

T was

all

creatine

CAT was

time

translocase

level ADP

of other

to

of

363

to

proteins

in

inhibit

label nucleotide

value

total

o [3H]ADP

the

to

be

equal

to

content

show

(18).

the

of

content

of

mole/mole

mitochondria Table

t 11e

binding

2.6 -2.9 in

per

incorporation

maximal

taken

estimations

or3H]ADP

binding

was

translocase

of

creatine

decreased

the is

and

dependent. and

16 moles

shown

),

CAT)

found

of

kinase

and

numerous

= T (Btot

presence

value

inactivation

This

( 17

- Beat

(T).

Both

carboxyatractyloside

translocase Btot

method.

about

incorporated.

,,,uM

mole

mitochondria

when

to

1

into

irreversible

incubation

binding

with

o[~H]ADP

and

inhibited,

o[ 3H]ADP

reflects

and

translocase

were

3

of

were

bound

) ).

filtration

incorporation 30-40

was

incorporation

a Millipore

After

of

( 16

TIiiE.flIN

of creatine kinase in The numbers show oADP 10 mW ADP was added; +ADP+ were added. The reaction “Material and Methods”. was 1.2 mg/ml , 21’C.

or3H]ADP

000

nucleotide

o[~H]ADP

mol

of

(M.W.=82 3a

determined

moles

, 40

30

Kinetics of inactivation mitochondria oADP. by concentration in q M. +ADP - 2aM ADP and 15 nM PCr PCr mixture iS given in Mitochondrial concentration

observed

of

20

1 shows

is T

Vol. 134, No. 1, 1986

0

BIOCHEMICAL

20

10

RESEARCH COMMUNICATIONS

30

TIME,MIN Pig.3.

AND BIOPHYSICAL

A - Kinetics of incorporation of o[~H]ADP into rat heart ritochondria. 1 - total incorporation: 2 difference between total incorporation and that in the presence of 50&M carboxyatractyloside; 3 difference between total incorporation and that in the presence of 0.5 l N ADP and 15 111 PCr. Inhibitor concentration in all cases was 0.7 eM. 30°C. 8 - Linear relationship between creatine kinase activity of rat heart mitochon ria and (ADP + PCr) B sensitive incorporation of o[ HIADP (see curve 3 in Pig.3A).

Table

1.

Translocase eitochondria,

and isolated

Cyt.aa naoles protein

creatine from different

kinase

contents. sources

activity, IU/eg

CK, mol/aol cyt .aa3

T, aol/Bol cyt .aa3

0.445i.o.09

4.5*0.5

2

35~0.25

2.65~0.25

0.353*0.07

3.0*0.3

2

43tO.26

2.70i0.2

2.9f0.3

2

23kO.19

2.80f0.20

0.354%0.06

0.86tO.2

2

60~0.34

2.90+_0.25

Rat liver aitochondria

0.246t0.04

0.00

0

00

2.60f0.25

,Rat heart mltoplast

0.360*0.06

2.5f0.3

2

02f0.3

2.3020.30

0.370*0.05

0.065iO.01

0.2OiO.03

2.36t0.30

Rat heart mitochondria Rabbit ritochondria

CK

? ag

heart

Dog heart mitochondria Chicken aitochondria

Rat

heart

heart

q itoplast incubated in KCl+ADP Mean

values

and

SD

are

given

for

364

four

experiments.

in

Vol.

134,

for

No.

BIOCHEMICAL

1, 1986

mitochondria

the

from

T/cyt.aa3 3a

Fig. which

completely

Fig.2)

decreases

in the

show

ratio

was

found

shows

also

that

the

.

of In

lower

ratio

is

Fig.

decrease

in

phosphocreatine mitochondria,

the

moles/mole

values

of

that

in if

treatment. content

determined

in

using

SH-reagents

chicken

heart

its

ADP

that

much

lower

effects

of

content the

for

work

( 20 1.

It

activity

is

types (Tabl.

Table

mitoplast

for

determination

kinase

+

to

Table was

membrane creatine be

highly

mitochondria determined

that to

in

value

of

in

notice content

mitochondria also

ADP

1.

same

proved

see

+

BCKmit)

from

cardiac

the

2.4-2.6

the

removed

1, 365

an ADP

the

of

oADP

additive.

in

kinase

a

When

= 0;

creatine

has

complete

as

interesting

mM

when

also

were

with

15

at

CKmit.

agrees

in

to

and

given

creatine

present

other

CAT

mitochondria of

BCKmit

CAT-treated

(determined

method

mitochondria

from

was

the

to

related

cyt.aa3.

value

was

taken

against

B CKmit.

kinase

was

( ATP

and

are

of3HIA”P

level

linearly

binding

is

and

translocase is

The

equilibrium

study

o[~H]ADP

cardiac

ADP

to

mitochondria

in

mM

an

used

contents

Thus,

,

This

1.

preparations

creatine

The

of

CK

liver

specific.

differ

on

mitochondrial

determined

kinase

The

3’

kinase 0.5

BCKmit

was

mixture

different

of

(see

mitochondria.

(BpCr,ADp

protect

that

maximal

cyt.aa

phosphocreatine

KC1

shows

kinase

phosphocreatine)

( 19 not

mixture

The

into

creatine kinase activity, max BCKmit. = 2.4-2.5 moles/mole

inactivation

by

does

phosphocreatine

+

establishes

low

mitochondria

creatine

creatine

kinase

T) 3b

to

all

ADP

of

presence

very

to

binding.

mixture

and

label

COMMUNICATIONS

constant.

= BCKmit ADP

creatine

affinity

1 shows

the

of

the

phosphocreatine, ADP/ATP

be

incorporation

the

RESEARCH

For

inactivation

presence

mitochondria.

the

to

- BPCr,ADP

binding

BIOPHYSICAL

sources.

prevents

Btot

binding

different

o[~H]ADP

difference,

AND

ref.

by that

in

does

not

in ( 21

spite )).The

Vol.

134,

No.

data

BIOCHEMICAL

1, 1986

of

Table

present

in

1 show

(Table

phosphocreatine In

shuttle conclusion

effectively proteins in

alterations creatine

for

of in

kinase

consistent

all

of

with

the

hypothesis

(or

oATP)

oADP

determination

of

the

content

may

be

mitochondrial

are cardiac of

). that

This

kinase

types

show

different

( 21

is

in

COMMUNICATIONS

creatine

results

mitochondria.

cases

This

RESEARCH

and

amounts

( 1,2,3 , the

used in

the

1).

BIOPHYSICAL

translocase

equimolar

almost

mitochondria

that

AND

method

pathology enzymes,

of

muscle including

of

ADP

especially which

can

be

binding important

may

translocase

involve and

).

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

21

V.A., Kupriyanov V.V., Elizarova G.V., Jacobus W.E. (1980) J.Biol.Chem. 255, 755-763. Jacobus W.E., Saks V.A. (1982) Arch.Biochem.Biophys. 222, 167-178. Saks V.A., Kuznetsov A.V., Kupriyanov V.V., Miceli M., Jacobus W.E. (1985) J.Biol.Chem. 250, 77577764. Riccio P., Scherer B., Klingenberg M. (1973) FEBS Lett. 3l, 11-14. DeMelo D.F., Satre M., Vignais P.V. (1984) FEBS Lett. 162, 101-106. Tsai P.K., Hogenkamp H.P.C. (1983) Arch. Biochem. Biophys. 226 --- 9 276-284. Johnson D., Lardy H.A. (1967) Methods Enzymol. 10, 94-96. Greenwalt J.W. (1968) J.Cell.BiolT-38, 158Schaitman C., 175. Van Gelder B.F. (1966) Biochim.Biophys.Acta 118 1 36-46. --(1976) Easterbrook-Smith S.B., Wallace J.C., Keech D.B. Eur.J.Biochem. 62, 125-130. Hansske F., Sprinzl M., Cramer F. (1974) Bioorg.Chem. 2, 367-376. Barbour R.L., Ribaudo J.. Chan S.H. (1984) J.Biol.Chem. 252, 8246-8251. Gerok W. (1983) J.Biochem. 94. 1247Blum H.E., Deus B., 1257. Meloche H.P. (1967) Biochemistry 5, 2273-2280. Milner-White E.J., Kelly I.D. (1976) Bi0chem.J. l..x, 23-32. Roberts R., Grace A.M. (1980) J.Biol.Chem. 255, 2870-2877. Klingenberg M., Appel M. (1980) FEBS Lett. 112, 195-199. Vignais P.V. (1976) Biochim.Biophys.Acta 456, l-38. (1977) J.Mol.Cell.Cardiol. 2, Altschuld R.A., Brierley G.P. 875-884. Kupriyanov V.V., Elizarova G.V., Saks V.A. (1981) Biokhimiia 930-941. 46, Bennet V.D., Hall N., DeLuca M., Suelter C.H. (1985) Arch. Biochem.Biophys. 240, 380-391. Saks

366