Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: Evidence for retarded dissociation of Ca2+ from the phosphoenzyme

Vol.

88, No.

June

27,

4, 1979

BlOCHEMlCAL

AND

8lOPHYSlCAL

RESEARCH

COMMUNICATIONS

Pages 1209-1216

1979

SLOW TRANSITION OF PHOSPHOENZYME FROM ADP-SENSITIVE TO ADP-INSENSITIVE FORMS IN SOLUBILIZED Ca*+, Mg*+ -ATPase OF S~.ACOPLASMIC RETICULUM : EVIDENCE FOR RETARDED DISSOCIATION OF Ca FROM THE PHOSPHOENZYME Yuichi

Takakuwa

and Tohru

Department Asahikawa Asahikawa Received

April

Kanazawa+

of Biochemistry Medical College 078-11, Japan

16,1979

SUMMARY: Solubilized Ca*+, Mg*+ -ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgC12. The phosphoenzyme formed was ADP-sensitive. Ca*+ in. the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCi2. These results showed that the transition was caused by dissociation of Ca2+ bound to the phosphoenzyme. Further observations indicated that, when Ca*+ in the medium was chelated, Ca*+ bound to the phosphoenzyme was dissociated much more slowly than Ca2+ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca2+- binding site in the phosphoenzyme. It

has been well

ulum vesicles

isolated

the vesicles. proposed

established from

ATP + 2Ca (out) (out)

Mg.E*P and phosphoenzyme

Ca*+ but not in random location tion,

i

for

Abbreviations bis(B-aminoethyl

& H20

-ATPase

catalyzes 2+

of sarcoplasmic

active

, Mg

2+

-ATPase

Ca2.EP (in)

--L

Mg.E*.Pi

which

have

: [32P]EP, 3*P-labeled ether)-N,N'-tetraacetic

EP e 2+ Pi + Mg (out> &E*~E

two binding

respectively,

E*P

sites

with

complex

inside

outside

takes

high

coupled

the vesicles.

affinity

combine

the membrane.

place

dephos-

2+

1, -

Ca*+ and ATP initially

the membrane

should

was previously

Mg

&

retic-

of Ca '+ by

uptake

2Ca*+ (in)

the Ca 2+ 1s released

correspondence

2+

E and EP represent,

to form Ca2 .E.ATP

of the Ca2+ across

To whom all

(1,2).

Mg

the Ca

Mg*+ on each molecule.

sequence

and then

muscle

ADP (out)

Ca2.E.ATP (out) (out)

EY

Ca *+,

scheme for

findings 2+

phoenzyme

skeletal

The following

from earlier

that

for with

Next,

to Ca *.EP

E

trans-

forma-

E" and E*P indicate,

be addressed. phosphoenzyme acid.

; EGTA, ethylene

glycol

0006-291X/79/121209-08$01.00/0 1209

Copyright Ail rights

@ I979

by Academic Press. Inc. in unyform reserved.

of reproduction

BIOCHEMICAL

Vol. 88, No. 4, 1979

respectively, with

dephosphoenzyme and phosphoenzyme which have one binding site 2+ but not for Ca2+ on each molecule. Our previous affinity for Mg

high

studies

(3)

demonstrated

that

enzyme.

The ATP formation

enzyme.

It

was concluded

from

of the step,

phosphoenzyme

can react

referred

added

transition

insensitive

form after

solubilized

Ca

2+

has been presented than

that

, Mg

2+

findings e

that

Ca2mEP + ADP.

Thus,

of phosphoenzyme

it

incapable in terms

EP, E*P and Mg.E*P

In this is

article,

referred

with

of sarcoplasmic that

reticulum

dissociation

Ca2eEP In the

form

vesicles,

ADP is

scheme,

ADP-insensitive.

from ADP-sensitive

when

to as ADP-

of reacting of the above

are all

resulted

to ADP-

Ca2+ in the medium has been investigated

chelating -ATPase

the ATP formation

ADP to form ATP,

one.

to indicate from

when ADP was added to phospho2+ when Ca was bound to the phospho-

phosphoenzyme

to as ADP-insensitive

study,

slower

these

while

whereas

only

Ca2*E*ATP

with

phosphoenzyme,

is ADP-sensitive, present

ATP was formed

occurred

from reversal

sensitive

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

and evidence

2+ of Ca from Ca2*EP is much

Ca 2.E. EXPERIMENTAL

[y-32P]ATP was prepared by the method of Glynn and Chappell (4). Sarcoplasmic reticulum vesicles were prepared from rabbit skeletal muscle as described previously (2), and lyophilized in 0.1 M KC1 - 0.1 M sucrose - 5 mM Tris-maleate buffer (pH 6.5). After reconstituted in the original volume of distilled water, the vesicles (0.5 mg of protein) were preincubated at 0' for 1 min in 0.43 ml of medium containing 2.5 ul of Triton X-100, 0.05 pmole of CaC12, 50 pmoles of KC1 and 50 umoles of Tris-maleate buffer (pH 7.0). No MgC12 was added to the preincubation mixture. They were solubilized within a few seconds, and so the suspension became clear. No membranous structures were detected on electron microscopic examination. Phosphorylation of Ca2+, Mg2+-ATPase in the sample was started by adding 0.05 ml of 1 uM [y-32P]ATP to. 0.43 ml of the solubilized vesicles, which contained contaminant Mg2+ at low concentration (lo-20 FM) in the medium (3). After adding various reagents as described under RESULTS, the reaction was quenched with 1 ml of 5 % perchloric acid containing 0.5 mM ATP and 0.2 mM Pi. The denatured sample was centrifuin the supernatant was extracted as phosphomolybdate complex ged , and [32P]Pi with isobutyl alcohol-benzene mixture, as described by Martin and Doty (5). [v-~~P]ATP was retained in the aqueous phase. Radioactivities of [32P]Pi and [y-32P]ATP thus isolated were measured by liquid scintillation counting. 32Plabeled phosphoenzyme ([32P]EP) in the pellet was determined as described previously (3). Protein was measured by the method of Lowry et al (6). RESULTS AND DISCUSSION Solubilized phorylated

with

Ca2+,

Mg2+ -ATPase

0.1 uM [v-~~P]ATP

of sarcoplasmic in the presence

1210

reticulum

vesicles

of 0.1 mM CaC12,

was phos100 mM KC1

Vol. 88, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1mM AOP al&

EGTA(O)

ttttttt

0

20 SECONDS

40

AFTER

60

ADDITION

OF [T-~~P]ATP

Fig.1. Formation of ADP-sensitive phosphoenzyme. Phosphorylation of solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum was started at 0' with [y-32P]ATP as described under EXPERIMENTAL, and 0.02 ml of 25 mM ADP - 25 mM EGTA was added at the time indicated by arrows. The reaction was quenched 6 set after adding ADP and EGTA (0). In the control, the reaction was performed without added ADP and EGTA under otherwise the same conditions as described above, and the reaction was quenched at the time indicated (0). [y-32P]ATP, [32P]Pi liberated and [32P]EP were measured as described under EXPERIMENTAL.

and contaminant

Mg2+.

phosphorylation

reached

sec.

On the other

responding ally.

attributable

inhibited

when the

increase

When a mixture [32 P]EP disappeared

in

to the turnover

( not

1, 20 set which

[ 32 P]EP, slow of

6 set

showed

The

[ 32 P]EP,

figure

by adding

40

a rapid

decrease,

cor-

it

declined

gradu-

liberation

was

was completely

excess

EGTA before

). after

[

32

and the corresponding

1211

reaction,

subsequent

[32P]Pi

because

the

for

and subsequently

rate.

was prevented

shown in the

starting

was maintained

of ADP and EGTA was added within

after

in the medium

at a very

[32 P]EP formation

reaction

level

[y- 32 P]ATP

hand,

was liberated

certainly

the

a steady

to the initial [32P]Pi

starting

As shown in Fig.

P]EP was formed, amount

of

most of 32 [yP]ATP

BIOCHEMICAL

Vol. 88, No. 4, 1979

1 ml.4

ADP

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

1mM

(*.A) JtttJ

1 mM

20

0

t

ADPi.,A) i

t

t

t

t

ADP(*.A)

60

40 SECONDS

AFTER

20

0

ADDITION

OF

40

60

[Y-~~P)ATP

(E) 100 a w a. 2 ? 5 ii-i z w u-l I k

6 .<

60

*,-

- - -A-

- ‘y

_

-m

-4

Chelating - -A

60 -

b

g*\

-

‘=.‘bw

.,,, o

40

*\

-.

. ‘\

.e

\

SECONDS

Fig.2. Slow transition forms induced by addition as described for Fig.1, 100 mM EGTA (A), 100 mM EGTA - 100 mM MgC12 (D),

t 0.2mM MqCl2 + 2 mM MgCl2

a

'\

0

4

concentration) EGTA EDTA EGTA EGTA

‘\ ‘4

1 2

2mM 2mM 2mM 2mM

-A

‘\

0

0 0 A A

\

0

20

reagents(final

(A) (B): (C) : (D).

‘.

L 6 AFTER

6 ADDITION

'\

't

0

10

12

OF CHELATING

REAGENTS

of phosphoenzyme from ADP-sensitive to ADP-insensitive Phosphorylation was started of chelating reagents. and 20 set later 0.01 ml of chelating reagents, i.e. EDTA (B), 100 mM EGTA - 10 mM MgC12 (C) or 100 mM was added as indicated by the first arrow (C). The

1212

8lOCHEMlCAL

Vol. 88, No. 4, 1979

was synthesized, results

whereas

clearly

indicating

of the

The ADP-sensitivity

of

measurement [32P]EP

( for

stated the

above,

but

reaction.

converted almost

set

indicating

2 (A),

was interrupted

of the

results

with

excess

transition [

Fig.

32

P]EP with

effects

inhibited

addition

of CaC12.

decreased

rapidly

is

clear Therefore,

was caused

reaction was was added at control, the time indicated phosphoenzyme given in (A

figure,

seems certain of Ca 2+ which

it

5 set

of that

set -1 .

quenched the time reaction

for

in place

after

transition

results

at least

20

2 (B) and (E), of EGTA,

2+ on this Mg of

kinetics.

These

was existent

transition.

[32P]EP

was induced

32 [ P]EP was converted of

[jLP]EP

addition

to

was almost

by subsequent of CaC12,

of [Y-~~P]ATP

[32P]EP

was bound

of

the transition

was reactivated after

amount

the start

first-order

the turnover

of EDTA, but

as

P]EP was gradually

As shown in Fig.

0.2 mM CaC12 was added

the ADP-sensitivity

by removal

2+

3, the transition

and the corresponding

it

Ca

of the contaminant

in Fig.

32

after

2 (E) shows that

to be 0.18

bound

When ADP was added

that

to the

of EGTA, and the ADP-sensitivity

when EDTA was added

As seen in the

by addition

[

one followed

was found

obtained

0.2 mM EDTA, and then

entirely

was bound

EGTA 20 set

addition

in 20 sec.

shown

form.

of

was initiated

the ADP-sensitive

one after

were

no appreciable

ADP-insensitive

Ca

the period

2+

phosphorylation

[32 P]EP to ADP-insensitive

In the experiment

CaC12.

in Fig.

to show that

similar

to be ADP-sensitive.

during

means that

Ca 2+ in the medium was chelated.

after

So it

This

ADP,

period.

disappeared

constant

evidence

with

).

state

retained

These

[ 32 P]EP and added

from

in the steady

As seen in the figure,

completely

quite

60 set

to ADP-insensitive

The rate

present

RESEARCH COMMUNICATIONS

became much slower.

[32 P]EP was entirely

given

it

from ADP-sensitive

give

[32P]EP

this

In the experiment

liberation

[y- 32 P]ATP was formed

at least

throughout

[ 32 P]Pi

the

show that

most

AND BIOPHYSICAL

[32P]EP

was synthesized.

was restored

by addition

to ADP-insensitive

to the ADP-sensitive

of

[ 32P]EP [ 32 P]EP.

6 set after 0.01 ml (A - C) or 0.03 ml (D) of 50 r&l ADP indicated by the second arrows (#) (A,.). In the was carried out without added ADP, and quenched at the (A,O). (E), the first-order kinetics of the ADP-sensitive after addition of chelating reagents was plotted from the data - D).

1213

BIOCHEMICAL

Vol. 88, No. 4, 1979

02mM

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EOTA

No Cd12

SECONDS

AFTER

ADDITION

OF

, No ADP

[d2PlATP

Fig.3. Restoration of ADP-sensitivity by addition of CaC12. Phosphorylation was started as described for Fig.1, and 0.01 ml of 10 mM EDTA ($), 0.01 ml of 10 mM CaC12 (&) and 0.01 ml of 50 mM ADP (t) were added in sequence as indicated by the arrows. The reaction was quenched 6 set after adding ADP (a). In the control, the reaction was performed without added ADP (X) or without added CaC12 and ADP (0) under otherwise the same conditions as described above, and quenched at the time indicated.

This

is

presence

quite

2+

of Ca

bound

The present

results

of ca2+ from cause lier.

consistent

it

took

with to

the previous

[32 P]EP is lead

In contrast,

20 set it

is

essential

to the striking

the ADP-sensitive about

evidence

r3* PJEP occurred for

evident

the transition that

for

(3)

that

the

the ADP-sensitivity.

conclusion

that

the dissociation

at an unusually to be completed,

Ca 2-b was dissociated

1214

indicating

slow

rate,

as mentioned very

rapidly

beear from

BIOCHEMICAL

Vol. 88, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

zz

5 0.03 i a r _ 0.02 aw E x ; 0.01 & f

2mM

EGTAIO)

0

-3

0

3

SECONDS

AFTER

6

ADDITION

9

OF

12

CT-32PlATP

Fig.4. Rapid prevention of phosphorylation by EGTA added just before starting the reaction. Phosphorylation was started as described for Fig.1 except that 0.01 ml of 100 mM EGTA was added 0.5 set before starting the reaction (0). In the control, the reaction was carried out without added EGTA (0).

the dephosphoenzyme vented

with

by EGTA which

in Fig. given

4. under

These

bound

was added

findings

from

Ca *E. 2

tion

from

the phosphoenzyme occluded

process tion

likely

in

might

(7).

Post

et al.

on the

As seen in sensitive

[32P]EP was added

transition

formation

Fig.

before

the observed be attributed

which

showed

of the occluded

was markedly

However,

slowed

that,

down

in terms

Ca2.EP

retardation

in

of the

formation

the

in the disso-

the transport

transition

from

mechasite.

the ADP-

by 0.2 mM MgC12

when 2 mM MgC12 was added

1215

of the

to Ca 2.EP forma-

binding

2 (D) and (E)).

scheme

is much slower than 2+ dissociain Ca

the cation

one was unaffected

( Fig.

of the

retardation

proposed

pre-

as given

of the enzyme during

similar

form

the rate

the reaction,

to be coupled

and originally

2 (C) and (E),

EGTA.

site

has proved

previously

was completely

to a possible

the Ca 2+ binding

to the ADP-insensitive with

starting

of Ca 2-t from

+ + Na , K -ATPase,

of K" from

nism based

(8)

phosphorylation

demonstrate

that

of Ca2+ translocation,

ciation

which

form

set

dissociation

that

transient

is

0.5

definitely

INTRODUCTION, It

Ca 2+ , since

with

These

EGTA, the results

indi-

BIOCHEMICAL

Vol. 88, No. 4, 1979

cate

that

also

was still

demonstrate

tivated

of Ca 2+ was not

the dissociation

dissociation

that being

by Mg2+,

more retarded direct again

The ADP-insensitive was quite

stable by only

is

that

evident

defferent reported (D),

from

hydrolysis

MgC12. observed

Further

with

2 min

the K+-sensitive, et al.

obtained

( not

in the present

study

by addition

shown

However,

as clearly

Indeed,

the

under

INTRODUCTION

chelating

reagents

in the medium. ).

shown

It

Accordingly,

insensitive

[32 P]EP was markedly

on the $-insensitive, is

of the

to K',

[ 32 P]EP which

ADP-insensitive

.

2+ of Mg . The results 32 [ P]EP was not ac-

in the figures

[ 32 P]EP was also

(9).

2+

the scheme given

100 mM KC1 was present

of the ADP-insensitive investigation

concentration

consistent

the ADP-insensitive

by Shigekawa

by high

by Mg

of the ADP-sensitive

[32P]EP

2 % for

accelerated

hydrolysis

at 0' even though

decomposed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was it

being

was previously in Fig.

2 (C) and

activated

by added

ADP-insensitive

[32P]EP

now in progress.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Kanazawa, T., and Boyer, P.D. (1973) J. Biol. Chem. 248, 3163-3172. Kanazawa, T. (1975) J. Biol. Chem. 250, 113-119. Kanazawa, T., Yamada, S., Yamamoto, T., and Tonomura, Y. (1971) J. Biochem, 70, 95-123. Glynn, I.M., and Chappell, J.B. (1964) Biochem. J. 90, 147-149. Martin, J.B., and Doty, D.M. (1949) Anal. Chem. 21, 965-967. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Sumida, M., and Tonomura, Y. (1974) J. Biochem. 75, 283-297. Post, R.L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540. Shigekawa, M., and Dougherty, J.P. (1978) J. Biol. Chem. 253, 1451-1457.

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