Rapid kinetic studies of calmodulin interactions with calcium and troponin I as monitored by anthroylcholine fluorescence

Vol. 106, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

June 30, 1982

RAPID

M.

I.

KINETIC STUDIES OF CALMODULIN INTERACTIONS WITH CALCIUM AND TROPONIN I AS MONITORED BY ANTHROYLCHOLINE FLUORESCENCE

Schimerlik-,

D.

A.

Department

Received

May

10,

1331-1339

S.

Malencik,

R.

Anderson

and

Y.

Shalitin

of Biochemistry and Biophysics Oregon State University Corvallis, Oregon 97331

1982

Anthroylcholine was SUMMARY: rapid kinetic studies of calcium and the calmodulin-troponin I lower than 70 nM, the mechanism scheme in which the dye binds of calmodulin and calmodulin-troponin also made ossible the estimation f lo* -M-1 S- ) and dissociation ing to the calciumq-calmodulin

utilized as an extrinsic fluorescent probe in dissociation from calmodulin (k,ff = 10 S-l) complex (k,ff = 6 S-l). At concentrations of dye binding agreed with the simple kinetic exclusively to the respective calcium complexes I. The sensitivity of anthroylcholine of values for the association((l.0 f 0.8) x rate constants (2 ? 170 S-l) for troponin I bindcomplex.

INTRODUCTION The often

second

messenger

mediated

The

by

binding

molecule

of

calmodulin

calcium

recognized

calcium

on

by

calmodulin

fluorescence

(3),

(5).

Recently

LaPorte is

fluorescence as

I and

We have in

*

stopped

flow

in

related

calmodulin

dependent

been

detected

circular

(6)

selectively and

displaced

cyclic

previously studies

To whom reprint requests the M. J. Murdock Charitable Grant H623632 and AM 13912 the recipient Of Research

by

used of

the

calcium

of (1‘2).

and

proton

that

the

fluorescence

the

calmodulin

The

effect

of includ-

magnetic

resonance probe

calcium-calmodulin calmodulin

(1,2).

measurements

(4),

the

is

proteins

spectroscopic

by

nucleotide

binding conformation

in

showed

bound

processes

proteins

dichroism al.

intracellular

calcium

a specific

--et

the

calcium

stabilizes

enhancement

troponin

of and

has

ing

anthroylcholine

role

9-

complex

binding

proteins

with such

phosphodiesterase.

intrinsic binding

and

correspondence Trust Grant of from the Muscular Career Development

tyrosine (7).

fluorescence This

report

of describes

should be addressed. the Research Corporation, Dystropy Association. Award HL 00796 from

calmodulin the

Supported by USPHS M.I.S. is NIH.

0006-291X/82/121331-09$01.00/0 1331

Cop.vrigh! 0 1982 by Academic Press, Inc. :I II righis o/ reproducrion in on,t form reserved.

Vol. 106, No. 4, 1982 application of

of

both

BIOCHEMICAL

9-anthroylcholine

calcium

and

AND BIOPHYSICAL to

troponin

I by

rapid

kinetic

RESEARCH COMMUNICATIONS

studies

on

the

binding

calmodulin.

MATERIALS

AND

METHODS

Kinetic studies were conducted using a Dionex D 137 stopped-flow photometer as described previously (4). Data were fit to equation (1) using Marquandt's algorithm as described by Bevington (8) by an LSI 11/23 minicomputer and the theoretical curve was then plotted superimposed on the experimental data. y = A0 t In equation equilibrium, amplitude of between

Al

e

-t/T

(1)

(1) y and ~ are the fluorescence intensities respectively. The observed exponential is All values reported Al and relaxation time -r. four and six experiments.

at time described are the

t and by average

Calmodulin was isolated from pork brain by the procedure of Schreiber (9) while troponin I was prepared by the method of Kerrick et al. (10). --et al. Anthroylcholine bromide was purchased from Molecular Probes, Inc. All experiments with calmodulin alone were done in a buffer composed of 20 m$ MOPS, while those with troponin I were done in the same buffer 0.15 M KCl, pH 7.2, plus i mM dithiothreitol. Excitation of the fluorescent probe was at 368 ? 3 nm using a 75 watt xenon lamp while emission was monitored using a Corning 3-72 cut-off filter.

RESULTS Our showed

previous

that

ment

the

(2.3

choline

stopped-flow association

msec, with

the

ing

anthroylcholine

the

binding

occurred

7).

the

Ca4

anthroylcholine cence

a single

anthroylcholine ential ing

as

observed

the

anthroylcholine

The was

difficult

time

(Fig.

dependence to

all

Ca

dye

determine

the

2+

dead

calmodulin

time on

of

the

mixing

with

a solution

results,

showing

-induced

was

change

mixed

the

lA),

in

of

EGTA

resulting the

(7-400

concentrations

on

with

When

determined

amplitude because

2t

to

instru-

anthroylcontainthat

both

calmodulin

had

domain.

concentration of

Ca

decay. was

the

similar

complex

exponential

for

the

binding

obtained

or

gave and

concentration

was

changes

calmodulin

-calmodulin

calcium

within

complex

concentration

followed

complete

-calmodulin

submillisecond 2+

on

fluorescence

anthroylcholine

the

When

2t and

of in

was

The

Ca4

studies

with

the

anthroylcholine

the

1332

non-linear

in

fluores-

dependence

of

T

the

increased.

constant

decrease

nM),

was

at

a single value

-1

expon-

of

T

-1

decreas-

concentration dependence

on

of

fluores-

Vol. 106, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1.1x

VOLTS

-0.27; TIME

IN

SECONDS

VOLTS

I

-0.22:

0.0410 TIME

Figure

1:

(A)

(B)

Calcium dissociation 1 mM Ca** plus was mixed with anthroylcholine. calculated from text, with -r-l

IN

SECONDS

from the (calcium)4-calmodulin 2 MM calmodulin plus 1.5 UM anthroylcholine an equal volume of 4 mM EGTA plus 7.5 The line through the data was the the fit of the data to equation (1) = 8 S-l, A, = .126 volts, Al = 0.640

complex. UM curve in the volts.

Troponin I binding to calciumq-calmodulin. 1 i;M calmodulin, 10 NM anthroylcholine and 1 mM calcium was mixed with equal volumes of 4 I.IM troponin I plus 10 u,M anthroylcholine. The line drawn through the data was calculated from the fit of the data to equation (1) in the text with -I-~ = 253 s-l, h = 0.37 volts, Al = 0.310 volts.

1333

Vol. 106, No. 4, 1982 cence

on

BIOCHEMICAL

concentration

factors,

at

determined

scatter

in

tudes.

the

The

using data

the

ence

of

. CaM

+ Ca4

binds

4 mol

T The

anthroylcholine

-1

= kl

(1 and

the

set

and

dye

at

the the

dye

S

-1

K = 41

part and

of

K = 62

completed

the

conditions calmodulin

agrees

+ 9AC 2+

- EGTA

experimental

conditions).

for

the

The

above

2B).

from

The

a single

plots

data

in

at

higher

complex

average

depend-

mechanism

is

(2)

calculated

of

the

class

of

of

Fig.

T versus

2B are

can

linear

bind

as

up

corresponds

sites,

we

is

many

constant

plot

anthroyl-

concentrations

dissociation

portion

which

dye

the

as

to not

4-6

440

UM

to

the

specific

kl

70 sur-

molecules

of

calculate

7 to was

the

calmodulin-troponin

concentration

+ 6 uM.

similar

the

11~4.

plot

(6).

= 10.1

(Fig.

? 0.7

dead

where concentration

the

50-80% time

which

troponin (Fig.

values

troponin

the

the

total

I concentration lB),

1334

0.4

of

anthroyl-

kl

= 6.2

mixed

resulted

was

UM calmodulin

in

pseudo in

The

a rapid

first

excess and

+ 0.4

with

change

Under

at

alone.

fluorescence

instrument.

with

phase

on

I was

concentration of

of

calmodulin

gave

T

-1

found

with

mixed

kinetic

of

2C)

in

a single

was

dependence

that

anthroylcholine with

showed

The

to

Experiments

a constant

I complex

also

400

reciprocal

decrease within

. CaM

the

-calmodulin

from

fluorescence

ampli-

-C 3 uM.

in

at

the

concentration

+ 4 Ca

under

be

an

concentration

calmodulin

order

2+

to

concentrations

linear

2+

2+

non-linearity

linear

a constant

choline

of

1 [anthroylcholinel/K)

K can

Ca

Experiments EGTA

the

treatment

concentration

with

that of

-1

kl

- CaMjCaM

(Fig.

anthroylcholine

binding

2+

The

since

Assuming

amplified

scheme

Ca4

+

PM anthroylcholine.

of

correction

k

concentration

prising

standard,

anthroylcholine

- 9ACs

Ca

on

constants

choline

on

kinetic

4 EGTA

-1

7

7

K

2+ Ca4

(Calmodulin

sulfate

Large

a quantitative

-1

RESEARCH COMMUNICATIONS

absorbancies.

prevented of

with

higher

a quinine

and

dependence

qualitatively

the

AND BIOPHYSICAL

either

of

BIOCHEMICAL

Vol. 106, No. 4, 1982

I aft

=-l

6-

(A

4-

AND BIOPHYSICAL

I

I

I

RESEARCH COMMUNICATIONS

I

I

I

*

I

(A) * ( * . *

+

2I

0

T

I

I

,

I

I

* I

I

_

!

,

/’

/’

/’

-

0.4

*

I

0.6-

(s)

.

t

* *

, /:

,-;

(B) I

0

I

I

I

1

I

_

I

I t

0.6

, xx’

-

-

*

, ,--’

(

//’ 0.4

-

,;

*A

CC)

0.2 n "0

I 50

I 100

(A)

Dependence Calmodulin, as in Figure

I 150

I 200

I 250

I 300

[9 Ant hroylcholine], Figure

2:

Dependence in Figure

(B)

of

on ~-1 EGTA and 1A.

of T on 2A replotted

?5OS limited

3.0 -1

,

reciprocal

are

the

same

Data

concentration. form

analysis S

and

of the linear < 70 PM) permitted K = 41 i 3 uM.

portion calculation

of

Dependence of i on anthroylcholine concentration for calcium dissociation from (calcium)4-calmodulin-troponin I. Data were obtained from experiments in which 4 UM calmodulin, 4 UM troponin I, 1 mM calcium, were mixed with an equal volume of 4 mM EGTA at varying anthroylcholine concentrations. Weighted least-squares analysis over the linear portion of the curve gave kl = 6.2 i 0.4 See-' and K = 62 i 6 PM.

~J.M troponin respectively.

since

concentration.

*1

Weighted least-squares the curve (anthroylchpline of kl = 10.1 Lt 0.7

or

I 400

pfvJ

concentrations

anthroylcholine in the

kl

2.0

I 350

[anthroylcholine]

T=1+

(Cl

anthroylcholine calcium

_

higher

I,

gave The

concentrations

values

for

conditions

T of

gave

1335

-1

the rates

of

218

+

33

s

experiment too

fast

-1

and

were to

measure

326

severely and

Vol. 106, No. 4, 1982 lower

BIOCHEMICAL

concentrations

determine.

The

data

gave

fluorescence

were

analyzed

.CaM.gAC+

2+

*CaM

Ca4

kl

+ TnIeCa4

k

the

pseudo

first of -1

T

Application 0.8

=

of

x

10'

the

simultaneous

rate

constant uM-40

equation

to

the

-1

mechanism

troponin

I

is

in

excess

and

K

the

two

k

=2+174S

-1 of

of

values

the

Control

independent

these

to

(3)

propagation

that

the

small

*CaM*TnI

where

and

equations.

to

too

+ 9AC

<<

+ k-1

s

changes

-1

[TN11

indicating

in

2+

conditions

-1

was

uM)

limiting

kl

this

from

*CaM

anthroylcholine

liters/m01

resulted

k-l

(5

order

concentration

2+

Ca4

RESEARCH COMMUNICATIONS

intensity according

K

2+ Ca4

Under

AND BIOPHYSICAL

of

T

-1 . errors

in

experiments dye

-1

The T -1

kl

= 1.1

large in

i

error

in

solution

of

showed

that

the

observed

over

the

range

studied

concentration

anthroylcholine

gives

dissociation

was

not

rate-

added

to

experiments.

DISCUSSION Observation choline

(varied

surprising however,

from

the

value

fit

the of

that

choline

nM could

the

fold

(6)

for

lower the

the

data

one

molecule

Fig.

mechanism lower

using

for

the

value

of

molecules

of

dye

of

was

more

dye

PM,

binding

bound tightly

data.

1336

sites

calcium

(10 (2)

with

Although (440

av

was

agreed

at

uM)

) calculated

with

the

fluorescence.

equation

(2)

found

saturation, bound

calmodulin

tyrosine

value by

is

IaPorte the

cannot

at

anthroyl-

constant

this

be

anthroyl-

rather on

-1

Set

a dissociation

dye. K

was

-calmodulin

intrinsic

70

the

2+

equation

agrees

than

EGTA

dye

for

2B to

kinetic

Ga4 of

constant in

when

plus

heterogeneity

(4)

calculated

phase uM)

determined

than 4-6

400

dissociation

the

be

kinetic

7 PM to

concentrations

ten

their

from

previously

Assuming

that

a single

considering

(6);

41

of

of about --et

al.

possibility ruled

out

by

Vol. 106, No. 4, 1982

The with

origin

(Ca

ing

3+

of

7 11M to

experiments

that of

absence

decrease

is

due

troponin

to

I has

significantly

different

calmodulin

niw)

troponin

I,

a further

decrease

interpretation Ca4

binding

2+

of

of 2+

Ca4

fluorescence

of

these

this

about

and

(Ca

the

sistent ent (Kd of the

2+ 4

the

binary

difference

uM)

and

dissociation

calcium

not

displaced

about

of of

by

troponin

sites

calcium

having

site I that

(Kd

complex. the

binding = 60

from

the

PM) -

kinetics

environments.

1337

4

-calmodulin data

that

Set

-1

(6 ) might

(Ca

-

complex are

from are

-1 also

also two

con-

differ-

present The

Set

2+ 4

conformation

I addition. complex

(10

Ca

arise

sites

the

constant in

These

troponin

ternary

complex

the

in

dissociation

difference in

and

to

dye

binding

The

con-

for

2+

simplest

a dissociation

site

a slight

the

bound

dependent

binding

reflect

dye I.

tight

in

of

The

the

the

increase

of

20%

anthroylcholine

the

with

EGTA.

a fraction

was

plus

excess

for

calcium-calmodulin in

to

(10

stoichiometry

site

anthroylcholine after

that

1:l molar

addition

was

mM)

binding

interpretation of

the

)

I

I agreed

amounting upon

-1

Set

troponin (0.5

fold

the

dye

(6

troponin

a two

in

bound

on

tight

-calmodulin)-troponin

populations

calcium

I.IM might

-1

the

anthroylcholine

with

= 40

also

at

found

T

of

confirming

affinity

I complex, 60

al.

of

calcium

with

anthroylcholine

highest If

uM)

decrease

was

dye

not

absence of

even

was

of

uM. was

\IM to the

--et

complex

the

62

40

experiments

fraction

the

Control

fluorescence

dissociation

titrations

However,

in

with

between

in

przI or

the

dependence

found

mixed vary-

with

calcium-calmodulin

the

was

interpret.

interact that

calcium

fluorescence

to

not

for

Laporte

authors.

-calmodulin

40

that

(10

of

calmodulin)-troponin from

dye

-calmodulin

heterogeneous stant

constant

experiments these

the

since

EGTA

concentrations

possibility of

discarded

when

difficult

I does

The

than

observed

more

concentration

plus

by

to

troponin

a rate

reported

total

free

RESEARCH COMMUNICATIONS

anthroylcholine

was

y - s t a t e fluorescence

(2

earlier

2C)

calcium.

been

gave

Stead

(Fig.

a low

concentration

the

decrease I at

FM

showed or

See-l).

fluorescence

400

presence

to

the

AND BIOPHYSICAL

-calmodulin)-troponin

4

from

BIOCHEMICAL

before slower

, compared reflect

rate to a

Vol. 106, No. 4, 1982 The origin troponin

of the

I was mixed

be due either in

constant

did

not

class that

displacement

high

(Kd = 40-60

affinity

a reduction

controlled

association decrease

faster

kinetic was poorly

value

was consistent

with

it

dissociation

from

quantitative

manner

was also

system

I; made

Ca4

the notion

at 9-anthroylcholin the

however,

the very

fast

analysis

2+

troponin

was near constant

to troponin

fluoresof

rate

con-

the low from

the

I.

involving

calcium

-calmodulin-troponin less

I in a than

70 uM.

of Ca4 2+ -calmodulin

interaction

and the

the for

total

dissociates

concentrations

rates

for

although

mechanisms

complexity

with

of the

difficult.

ACKNOWLEDGMENTS The authors would like to acknowledge Dr. S. G. Blanchard and Rosemary Owen for help with the computer programming, Paula Sparks and and The Murdock Charitable Trust Sue Conte for typing the manuscript, for a grant to purchase the rapid kinetic equipment. 1338

a

this

the presence

calcium prior

expect

be expected

The dissociation

and Ca4

to study

rate

measurements

range

competitively

studies

experiments,

that

rate

the possibility

constant

these

inter-

the

one would

20 to 50% of the

to treat

-calmodulin

over

50% would

bimolecular

complex

observed

calmodulin

rate

only

from these

possible

a thorough

binds

be discounted.

was possible 2+

site

the

in these

cannot

determined

for Since

I-(calcium-calmodulin)

In summary,

troponin

(11)

was observable

stant

troponin

limit

phases

preclude

from

Both

K = 440 uM for

data

estimated

the

binding

The association

of dye may

sites.

was varied

r -1 of about

in

of two molecules.

cence

It

These

to calcium-calmodulin

diffusion

that

when

I or to a decrease

at peripheral

one assumes

anthroylcholine

uM) since

the presence

observation

10% if

sites.

at 40 uM dye concentration. binding

the

than

found

of dye by troponin

case of competitive

of less

of anthroylcholine the

intensity

when dye concentration

In the

RESEARCH COMMUNICATIONS

in

of dye bound with

change

in rate

in fluorescence

calcium-calmodulin

consistent

5 uM to 40 uM.

reduction

with

enhancement

were

AND BIOPHYSICAL

decrease

to direct

fluorescence

pretations

of

BIOCHEMICAL

I

Vol. 106, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES

1. 2. 3. 4.

5. 6. 7. a. 9. 10. 11.

Cheung, W.Y. (1980) Science 207, 19-27. Means, A.K. and J.R. Dedman (1980) Nature 285, 73-77. Dedman, J.R., J.D. Potter, R.L. Jackson, J.D. Johnson and A.R. Means (1’377) .J. Biol. Chcm. 252, 8415-8422. Klee, C.B. (1377) Biochemistry 16, 1017-1024. Crouch, T.H. and C.B. Klcc (1980) Biochemistry 19, 3692-3698. LaPorte, D.C., B.M. Weirman, and D.R. Storm (1980) Biochemistry 19, 3814-3813. Malencik, D.A., S.R. Anderson, Y. Shalitin, and M.I. Schimcrlik (1981) Biochem. Biophys. Res. Comm. 101, 390-395. Bevinyton, P.R. (1969) Data Reduction and Error Analysis for the Physical Sciences (Chap. 11) McGraw-Hill, N.Y. Schreiber, W-E., T. Sasagawa, K. Titani, R.D. Wade, D.A. Malcncik and E.H. Fisher (1981) Biochemistry 20, 5239-5245. Kcrrick, U.G., D.A. Malencik, P.E. Hoar, J.D. Potter, R.L. Colby, S. Pocinwing and E.H. Fisher (1980) Pfliigcrs Arch. 386, 207-213. Ilammcs, G.G. (1978) Principles of Chemical Kinetics, Academic Press, M.Y., 11. 6 5 .

1339