The activation of rabbit skeletal muscle phosphorylase kinase requires the binding of 3 Ca2+ per δ subunit

vol. 105, No. 2, 1982 March 30, 1982

THE ACTIVATION

BIOCHEMICAL

OE' RABBIT

SKELETAL

BINDING; Danielle

BURGER,

Department

of

Jos

January

A.

3 Ca

COX,

Biochemistry,

4,

MUSCLE

OF

2+

PER

Edmond

H.

8,

KINASE

REQUIRES

THE

6 SUBUNIT FISCHER*,

of

Geneva

RESEARCH COMMUNICATIONS Pages 632-638

PHOSPHORYLASE

University 1211

Received

AND BIOPHYSICAL

Eric

Geneva,

P.O.Box

t

A.

STEIN

78

Jonction,

Switzerland

1982

SUMMARY : Rabbit skeletal muscle phosphorylase kinase (EC 2.7.1.38) binds 12 G12+, i.e. 3 per 6 subunit; among these three &a-binding sites, one iias a high affinity (Kdiss = 0.31 iiM) and two have a lower affinity (Kdiss = 2.15 LiM). Thus, the binding of calcium to the enzyme-bound calmodulin, called 6 subunit, is different from the binding to free calmodulin. The activation of phosphorylase kinase occurs when three Ca 2+ are bound to the ri subunit. Rabbit

skeletal

uNw4

(1).

which

directly

identical the of

with

was

enzyme

was

were

bovine

modulin

* t

out brain

an

Department To whom all Abbreviations:

(2).

(3)

and

claimed

there

is

2t

in

as

intrinsic of

4,

The

G),

to

for

investigate the

2t

of

been

-binding

the

shown

and

different Ca

2+

calcium, to

subunit

capacity

of affinity

-binding

to

phospho-

a relationship

calmodulin-mediated of

adenylate

this

correlation

only

known

enzyme

acticyclase

in

the

which

(8).

Inc. reserved.

632

It

instance

of

contains

cal-

University of Washington, Seattle, WA, USA. Biochemistry, correspondance and requests for reprints should be addressed. TES, N-tris(hydroxymethyl)metilyl-2-aminoethanesulfonic ethyleneglycol-bis(R-aminoethyl ether)-N,N,N',N' acid; EGTA, tetraacetic acid; HEDTA, N'-(2-hydroxyethyl)-cthylenediamine N,N',N'-triacetic acid.

0 1982 b-v Academrc &es& of reproduction in any form

be

ionic

Such

lacking. the

and

structure

subunit.

0006-291X/82/060632-07$01.00/0 C’opyrrghl All righis

is

(7)

is

Ca

between

laboratory

latter

the

has

because

activity

subunit

concentration

about

a correlation

to the

be

apparently

-dependent

the

h subunit

disagreement

5,

this

has

micromolar

phosphodiesterase

interest kinase

as

protein

(2,

Ca

kinasc

requires

Moreover the

particular

phosphorylase

the

2+

used.

worked

of of

to

Ca

and

recently

activity

However for

kinase

vation

enzyme

phosphorylase

calmodulin

(4).

strengths rylase

The binds

enzyme the

muscle

BIOCHEMICAL

vol. 105, No. 2, 1982

MATERIALS

AND BIOPHYSICAL AND

RESEARCH COMMUNICATIONS

METHODS

Proteins. Rabbit skeletal muscle qlycogen phosphorylase :, was purified by the (9) and stored at -20° C in 60 mM TES, pH 7.0, li mM procedure of Krebs c.: :1. mercaptoethanol, 5OY (w/v) glycerol. Non-activated phosphorylase kinase was prepared from rabbit skeletal muscle The specific activity of the enzyme measured as reaccording to Cohen (10). commended by Shenolikar ,~i .;i. was 10 i 2 U/mg at pi 8.2, and the activity Cl), ratio at pH 6.8 over pH 8.2 was 0.008 ? 0.002 (mean of 5 different preparations). C in 50 mM glycerophosphate, The enzyme (,-,-i 40 mg/ml) was stored at -20° pH 7.0, 2 mM EDTA, 15 mM mercaptoethanol, 50% (w/v) glycerol. The enzymes were homogeneous as seen by sodium dodecyl sulfate-polyacrylaProtein concentrations were determined by ol,mide gel electrophoresis (11). tical density, using absorption coefficients, A,1 I of 13.1 for phosphorylase 280 ml (12) and 12.4 for phosphorylase kinase (10). The assay mixture (100 ~1) Ca2+-dependent activrty of phosphorylase kinase. included GO mM TES, pH 7.0, 6 mg/ml phosphorylasc !', 1.61 ug/ml phosphorylase kinasc, 50 lig/ml bovine serum albumin, 4.5 mM mercaptoethanol, 10‘; (w/v) glycerol 3.6 IBM ATP, 12 mM Mgcl . Free Ca2+ concentrations were controlled as described below. The reaction w& carried out at 30° C and initiated by the addition of ATP and MgCl2. After 5 min, 10 1~1 aliquots were diluted 20-fold with ice-cold 0.1 M sodium maleatc, pH 6.5, 15 mM mercaptoethanol, 1 mg/ml bovine serum albumin, and assayed for phosphorylase :i according to Hedrick and Fischer (13); 70 ~1 aliquots were diluted lo-fold with a 0.5': (w/v) EDTA solution (14) to measure the total Ca2+ concentration. Ca2+ binding to phosphorylase kinase. The binding was measured by gel filtration according to Hummel and Dreyer (15). A column (31 x 0.9 cm) of Sephacryl S-200 was e uilibrated at 4O C with 60 mM TES, pH 7.0, 8.0 mM MgCl2, 1 to 2 LICi/ml 4'Caqf. The free Ca2+ concentrations were controlled as described below. 'Two to three mg of phosphorylase kinase diluted in 200 ~1 of the column buffer were applied to the gel and eluted at a rate of about 120 \ll/min. Fractions of 1 ml were collected and measured for phosphorylase kinase concentration. The concentration of bound Ca 2+ was measured by liquid scintillation counting. Neither the specific activity of phosphorylase kinase nor its subunit pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were changed by the chromatography. Total jCa*+/ was measured after a two-fold dilution with bidistilled water. The amount of bound Ca 2+ was calculated per 1.3 x 106 g protein (10, 16). The data were analyzed by a nonlinear least-squares curve-fitting procedure witia computer program. 2+ Ca2+ -concentration measurements. In both Cd -binding experiments and activity tests, free ICaL+ was contrqiled by means of EGTA (1 mM or 0.2 mM) or HEDTA (1 mM or 0.2 mM). Total ICa 1 was measured by atomic absorption using Titrisol standards (Merck, Darmstadt, Germany) diluted in the same conditions as the sample, i.c. in 0.5% (w/v) EDTA or 9% (w/v) glycerol. Free metal ion concentrations were calculated with a computer program adapted from Perrin and Sayce (17) taking into account the interactions of H+, Ca 2+ and Mg2+ with EGTA, HEDTA and ATP. For these calculations the association constants of Martell and Smith (18) were used for complexes with EGTA, those of Cohen (4) for complexes with HEDTA, and those of Donaldson and Kerrick (19) for complexes with ATP. The distribution of the complex species S.Ca,, incorporated in the holoenzyme, was calculated as a function of free jCa 2+;as previously described (7). RESULTS Calcium

dependence

pnorylase

kinase

of by

phosphorylase free

Ca

2-t

kinase occurs

in

activity. the

633

-

micromolar

The range

activation (Fig.

of 1).

phosThe

BIOCHEMICAL

Vol. 105, No. 2. 1982

Figure

free

calcium

(n=6). of

be

per

kinase

in

terms

of

of 100

the PM

those

6 PM and Comparison intrinsic

stoichiometric

2+

as of one

for

Ca

6 is

binding

of

Ifree.

Hence

6 subunit

in

21

of

a Kdiss

between

calcium

binding constants

2+

much

phosphorylase the

binding

constants were

UM

3 Ca

buffers

2+

are

-binding

could a disof

drss

existence

of

demonstrated

containing of

more

calmodulin

definitely

distinct

sites

with

a Kdiss

of

(7). and

phosphorylase

obtained calculated

634

by

computer

as

previously

kinase

2+

phosphorylase

be

properties

kinase

has 200

in

not

Ca

with a K

The

could

calcium-binding

phosphorylase

of

observed.

kinase

sites

of

12

data

with

subunit

affinity

1).

The

sites

k 0.6

plot

binds

3.9

8.4

3.8

Hill

Fig.

2).

sites:

d is

The

(inset

(Fig.

PM and

per

lower

calmodulin,which

with

curve

0.31

.

kinase

calcium-binding

3 Ca

with

= 1.1

independent

the of

site

free

of

2+

PM Ca

Phosphorylase

saturation

types

aggregation

/Ca

the

H

is

0.5’

0.1 n

kinase.

two

that

below

The enzyme represents

inset

jCa 2+ 1

activation,

a coefficient

by

(Kdiss)

a maximal

incorporated

the

shown

Ca2+ -binding

because

maximal

inactive

yields

RESEARCH COMMUNICATIONS

ph;sphorylase kinase activity. Ca 1s taken as loo%. The Hill plot (an = 1.1).

half

phosphorylase

as

cl),

of !.IM

virtually

Considering

a fourth

from

to

constant PM.

2.15

is

(n&6)4

analyzed

at

curve

binding

sociation

than

enzyme

activation

Calcium ions

Ca -dependence activity at 100 the corresponding

concentration

The

the

2+

1.

AND BIOPHYSICAL

From

activity. ~___

analysis, described

the

three (7);

they

PM

BIOCHEMICAL

Vol. 105, No. 2, 1982

Figure

equal

4.18

pectively.

Calcium binding Scatchard plot curves calculated free Ca2+ (PM).

x 10' From

calculated was

2.

and

obtained

M

-1

, 1.16

these

the

with

3.

solid

-1 M

2.17

x 105

percentage

of

105

6-Q

3

activity

species.

Distribution of the total amount A is the distribution 6.Ca3 and C of phorylase kinase of B (n = 1.4). kinase &tivity

and

the the

I3

Figure

to phosphorylase The (inset). by computer.

constants,

compared for

x

AND BIOPHYSICAL

1

kinase (PhK) and the corresponding lines represent the theoretical b/f is the ratio of bound Ca2+ to

(Fig. The

small

4ogKa 62* I

the of

RESEARCH COMMUNICATIONS

M

-1

each The

3). difference

for

K

6.Ca best

tc2 and

1'

species

n

K3 reswas

correlation

between

the

two

5

6.Ca complexes (expressed in percentage of 6 asna function of free Ca2+ concentration. of 6'Ca + 6.Ca + 6.Ca B of 6.Ca + 1 2 3' 'Ca3. The dashed curve is the activity o z phosas in Figure 1. The inset shows the Hill plot c (n, = l.l), the dashed line the phosphorylase (n = 1.1). H

635

Vol. 105, No. 2, 1982

BIOCHEMICAL

curves

is

of

good

agreement

vity

(n

eluded

in

the

range between

= 1.1)

H

that

and

of

the

experimental

the

Hill

the

S'Ca3

([email protected]

is

AND BIOPHYSKAL errors.

coefficients

the

RESEARCH COMMUNICATIONS

of

species

(nH

active

Furthermore the

of

is

calcium-dependent

= 1.1).

species

there

Hence

it

actican

phosphorylase

a

be

con-

kinase.

DISCUSSION At

the

zymatic of

apprently

for

Mg

into

of

2+

, Ca

2+

all

buffer,

is

2+

ofMg 0.3

PM)

the

K

class

-

(5), These

and

is

about

not

the

treatment

required

when

of

action

of of

tween

the

those

of

the

data

the

2+

same

same

6 subunit

of

with

the

Ca

here

are

properties 2+

that

with

not

6.8

high

affinity

a lower

affinity

number

of

two

1,

in

this

to

-binding have

those

Such

independent

classes

where

experi-

of in

Kilimann

the

presence

sites

(Kdiss

= 2.9

PM).

Although

sites

within

each

disappeared

performed.

was

different.

strength

(Kdiss

yM

study

significantly

ionic

2+

7.3

the

Ca 2'-binding

Ca

(20) taking

s of

in

identical

might

been

2+

differences

high

those pH 6.8,

recalculated

ICa

not

en-

glycerophosphate

found

are

at

between

the

-binding

disagree

and

a

the

if

a mathe-

an analysis of

=

is

sites

indeed

with

rather

(21).

observed shows

results

possesses

of

were

the

both

had

-dependence

buffer,

(19)

23 PM,

than

discrepancy

constants

calmodulin Ca

this the

of

obtained

the

a protein

which

those

with

same;

difference

modulin,

found

similar,

dissociation The

were

4 sites

very

matical

close

which

ATP

Considering that

2+

here

results

higher

presented

authors

are

drss

feel

data

of

Instead

used.

Ca

a Tris-glycerophosphate

Cohen's

two-fold

was

the presented

affinities

chelates.

we

Heilmeyer

used

Therefore

only

conditions, binding

concerning kinase

the

the

pH 7.0,

The

data

author

+ H .

and

which

obtained,

and

latter

disregarded

account

mental

the

phosphorylase

The

(4).

and

and

glance,

activity

Cohen

TES

first

-binding

the

affinity

Ca for

phosphorylase catalytic of protein

2+

-binding

its

three

kinase y

a free as

636

part

high may

subunit Ca

parameters

(22, 2+

affinity

be

due

a protein

free

cal-

sites, to

23).

-binding

of

of

the

inter-

Differences

protein complex

be-

compared have

to al-

BIOCHEMICAL

Vol. 105, No. 2, 1982 ready

been

described

affinity

for

troponin

complex The

(8)

calcium

in

is

achieved TO conclude,

calcium but

when

still

one

the

C:

order

of

latter

RESEARCH COMMUNICATIONS

also

magnitude

shows

when

an

increase

incorporated

in

in the

(24). of

this

activities

troponin

of

activation

obtained dent

for

AND BIOPHYSICAL

phosphorylase

1aborator.y

for

of

bovine

by

calmodulin*Ca3. this

the

other

brain

work

incorporated

requires

kinase

6.Ca

enzymes:

that

of

calmodulin

at

of and

as 2+

3 Ca

an

for

the

of Ca

adenylase

changes

kinase least

reminiscent

regulation (7)

phosphorylase

binding

is

3

phosphodiesterasc

shows in

by

its

2+

-depen-

cyclase

affinity

intrinsic

the

results

for subunit,

activation

of

the

enzyme.

ACKNOWLEDGEMENTS The Theorique work

was

authors

are

grateful

Appliyuee, supported

to

University by

the

Dr of

Swiss

J. -J.

Combremont

Geneva)

NSF

for

grant

No

(Laboratoire

the

computer

de

Chimie

programs.

This

3.237.77.

REFERENCES

t. 2. 3.

Shenolikar, S., Cohen, P.T.W., Eur.J.Biochem. 100, 329-337. Brostrom, C.O., Hunkeler, F.L., 1961-1967. Grand, R.J.A., Shenolikar, S.,

Cohen

P.,

and

Krebs,

and

Cohen,

Nairn, E.G. P.

A.C., (1971) (1981)

and

Perry,

S.V.

J.Biol.Chem.

(1979) 246,

Eur.J.Biochem.

113,

!??I-367.

~I.-' -3. -4.

Cohen, P. (1980) Eur.J.Biochem. 111, 563-574. Kilimann, M., and Heilmeyer, L.M.G., Jr. (lY77) Eur.J.Biochem. 73, 191-197. Ozawa, E. (1972) J.Biochem. 71, 321-331. Cox, J.A., Malno&, A., and Stein, E.A. (1981) J.BioL.Chem. 256, 3210-3222. Malnoe, A., Cox, J.A., and Stein, E.A. (19UI') Bioc!iem.Biophys.Acta 714, in prl Krebs, E.G., Love, D.S., Bratvold, G.E., Trayser, K.A., Meyer, W.L., and Fischer, E.H. (lCJG5) Biochemistry 3, 1022-1033. Cohen, P. (1973) Eur.J.Blochcm. 34, l-14. Lamml~, U.L. (1970) Nature '27, 680. Cohen, I'., Ducwcr, T., and Fischer, E.H. (1971) Biochemistry 10, LGdU-2~94. Eiedrick. J.L., and Fischer, E.H. (1965) Biochemistry 4, 1337-1341'. Ramakrishna, T.V., West, P.W., and Robinson, J.W. (1968) Anal.Chim.Acta 40,

~':. .//_ ~'7.

Hummel, tlayakawa, Perrirl,

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J.P., T., D.D.,

and Dreycr, W.J. (1062) Biochim.Biophys.Acta 63, Perkins, J-P., and Krebs, E.G. (1173) Biochemistry and Sayce, I.(;. (1907; Talanta l,!, Ui3-642.

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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