Calcium-dependent interactions of an ionophore A23187 with calmodulin

Vol.

130,

No.

July

16, 1985

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

CALCIUM-DEPENDENT Masaki

INTERACTIONS

Inagak

, Toshio

Department

Received

May

of

22,

OF AN IONOPHORE

Tanaka,

Yasuharu

Pharmacology, Edobashi,

A23187

WITH

CALMODULIN

and

Hiroyoshi

Hidaka*

School

of

Sasaki

Mie University Tsu 514, Japan

ZOO-206

Medicine,

1 985

We found that ionophore A23187 interacted reversibly with calmodulin in a calcium-dependent fashion. It was found that A23187 interacts (CaM), selectively with CaM, among calcium binding proteins (such as troponin C However, apparently differing from and S-100 protein) and other proteins. W-7, A23187 did not supprF?s CaM-dependent enzyme activity such as myosin -dependent cyclic nucleotide phosphodiesterase. light chain kinase and Ca Our observations suggest that there are novel calcium-dependent regions of CaM which can be monitored using ionophore A23187 and may not be related to enzyme activation, Q 1985 Academic Press, inc.

The

polyether

examine

the

studies

have

spectrum

antibiotic

regulatory

of

biological

control

ubiquitously

physiological

A23187 ionophore action

on

report

the

0006-291X/85 C’ofivrlghr All rights

on

have

whom

for

involvement

the

eukaryotic for

a wide

of

Ca 2t

in

muscle

to These a wide

contraction,

gluconeogenesis,

the

other

hand,

cells

and

has

been

calcium

ion

and

the

of

cellular

range

widely system.

fertilization, On

used

biological

including

(1,2).

receptor for

in

now

calmodulin

is

proposed

as

a

major

responses

evoked

by

(3).

Studies

To

in

effector

cation

ion

others

distributed

is

calcium

mitosis,

many

intracellular

A23187

mechanisms

coupling, and

universal

of

evidence

glycogenolysis,

*

role

provided

stimulus-secretion

the

ionophore

the

mechanism

traditionally and

lipid

the

Ca

evidence

all

and focused

bilayers.

2+ -CaM

on

the

the

system Ca 2+-dependent

correspondence

and

the

Inc. reserved.

biological

has

reprint

200

effects

between molecular not

the

been

requests

of

calcium

mechanisms

interaction

$1.50 (8 19x5 by Academic Press, of reproduction in anv .form

of

interactions

However,

regulatory

for

specificity

of

established. of

should

A23187 We now

A23187

be

with

addressed.

CaM.

Vol.

130,

No.

BIOCHEMICAL

1, 1985

AND

MATERIALS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

A23187 was obtained from Calbiochem-Behring Corp. [y -32PJATP was The myosin light chain kinase from obtained from Amersham, England. chicken gizzard was purified by the method of Adelstein and Klee (4) Calmodulin-deficient myosin light chain of chicken gizzard, used as and substrate for the kinase assay, was prepared by the method of Perrie Perry (5). The light chain was separated from cal?qdulin by DEAE-ce lulose e from (Whatman) chromatography. Calmodulin-deficient Ca -phosphodiesteras bovine brain and bovine brain calmodulin were prepared as described ( 6). Protein was measured by the method of Lowry et al. (7) with $gvine se rum Ca"-dependent albumin or purified bovine brain calmodulin as standard. cyclic nucleotide phosphodiesterase was measured using our own method (8). CaM was assayed for its ability to activate a fixed amount of CaM-deficient phosphodiesterase under standard conditions (9). One unit of CaM was defined as the amount activating 50% of the maximal phosphodiesterase activity attainable under standard experimental conditions, and was equivalent to long of CaM (9). The equilibrium binding procedure between [ H]W-7 and CaM followed that described by Hummel and Dreyer (10). Myosin light chain kinase activity was jfsayed in 0.2 ml of 20 mM Tris-HCl 20 pm [YP]ATP (3lrCilassay tube), 35 PM smooth (pH 7.0), 10 mM MgCl muscle myosin light $iain and either 0.05 mM CaCl or 2 mM EGTA at 30°C. Unless otherwise noted, 0.2 pg calmodulin was use ii . Rates of phosphate incorporation were constant during the incubation period. For some experiments, incubations were terminated by addition of 20 r.ll of 10% sodium dodecyl sulfate with 5 ~1 of 100% mercaptoethanol. Samples (60 ~1) were subjected to electrophoresis in 15% polyacrylamide containing 0.24 SDS. Gels were either stained with Coomassie brilliant blue or divided into 2-mm slices for liquid scintillation counting. Alternatively, incubations were terminated by the addition of 1 ml of 10% trichloroacetic acid followed by centrifugation (3000 rpm, 10 min). The pellet was suspended in 5% trichloroacetic acid and the centrifugation-resuspension cycle was repeated three times. The final pellet was dissolved in lml of 0.5 M NaOH for radioassay. Phosphodiesterase activity was assayed as previously described. Smooth muscle cells, derived from medial layers of the rabbit thoracic aorta were prepared according to (11). After careful removal of the adventitia, medial rings (thickness 0.5 mm) were placed on Falcon flasks and cultured in a MEM medium supplemented with 106 calf serum. After 3 weeks, growth of primary cells around the explants ceased. Cells were then trypsinized and dispensed into flasks for subculture. The cells were incubated at 37°C in 5% CO and the medium was changed every 2 days. Smooth muscle cell culture $ exhibited at confluency the characteristic "hills and valleys" pattern (12). Electron microscopy was routinely performed. A23187 was applied to cell preparations at a concentration of 1 pM in phosphate-buffered saline for a period of 2 min at 37°C followed by rinsing with phosphate-buffered saline and the preparations were examined using an Olympus microscope (BH-2) equipped with an epifluorescence apparatus and UV filter was used for fluorescence microscopy. Photographs were taken using Kodak Tri-X pan film. Fluorescence spectra were recorded using an Aminco-Bowman spectrofluorometer with a thermostat at 20 f 1°C. Total dilution never exceeded 5% and relative fluorescence values were uniformly corrected for dilution. A23187 was dissolved in 100% dimethyl sulfoxide and added so that the concentration in the samples undergoing measurement was 0.52. Excitation was at 350 nm or 280 nm and fluorescence intensity was monitored at 437 rim. Solutions contained 50 mM Tris-HCl (pH 7.5), 0.1 mM. RESULTS AND DISCUSSION The fluorescence its

interactions

with

of A23187 the

target

provides molecule.

201

a convenient Figure

means for 1 suggests

examining that

Vol.

130,No.

1, 1985

BIOCHEMICALAND

500 450 Wavelength

BIOPHYSICAL

400

RESEARCHCOMMUNICATIONS

-7 -6 Cd*Concentration(Log

-5

-4 M)

of calmodulin on A23187 fluorescence. spectra of 3 pM A23187 iF+50 mM Tris-HCl (pH 7.5) with or without 15 IJM calmodulin, and 50 fl Ca . Excitation at 350 nm. (6); (0); Solutio?? contained 50 mM.Tris-HCl (p;xzi;A;ibi 2 ;;Am;:lin, 3 pM A23187 and Ca -EGTA bufser as indicated. emission at 437 nm. (A); [ HN-7 binding to calmodulin was measurgd as described under Experimental Procedures.

ionophore

A23187

A23187

in

maximum

an aqueous

at

456

significant in

maximum

ethylene

glycol

did

affect

with

nm.

In

not

to bis

to

the

of

regions

of

increased

CaM with

concentration

A23187

in

The

1 PM calcium

ion

for

of

CaM

Ca

(Fig

2+

by

was

CaM

is

of

related

in

acid

(EGTA),

alone

can

was

mM

did

not

serve

as

an

significantly

with

10 ~J-M Ca

enzyme

activation

to

that

polar

media

Ka 1 mM (14), to

(2

2+

.

The and

Ca*+ Ca

2+

N-(6-aminohexyl)-5-chloro-l-

similar

A23187

ions

hydrophobic

fluorescence

as

shift

nm.

-dependent

maximal

such

ion

be

2+

, CaM-dependent

As

1B).

calcium may

and

ions

environments

Ca

A23187

antagonists

(W-7)

for

of

437

a

a blue

calcium

Calcium

nonpolar

with

produced with

of

at

fashion.

spectrum CaM

absence

A23187

into

effect

-dependent

intensity

the

of

partitioning

(13).

fluorescence

In

fluorescence

ionophore

affinity

1A).

enhancement

fluorescence

binding

fluorescence

enhancement.

naphthalenesulfonamide A23187

mM CaC12,

a significant the

binding

0.1

2+

emission

ether)-N,N,N',N'-tetraacetic

required

dependent

(Fig.

a Ca

a broad of

A23187

in

(B-aminoethyl

fluorescence

indication

presence

nm

significantly This

CaM,

displayed

in

437

lead

purified

solution

enhancement

the

CaM

interacts

this

Cazf-binding

to

needed at

to

increase

neutral

pH has

enhancement CaM

and

a

in not

to

A23187. Table calcium

I shows binding

the

proteins

influence and

of several

calcium

ion

other

proteins.

202

on

the

binding Calcium

of ion

A23187 had

to a

Vol.

130,

8lOCHEMlCALAND8lOPHYSlCALRESEARCH

No. 1, 1985

Table Interaction

of

COMMUNICATIONS

1

A23187

with

various

Increase of fluorescence Proteins

proteins relative intensity*

0.1 lnv CaC12

2 mM EGTA

53.0 4.8 3.5

0.0 0.0 0.0 0.0

Calmodulin S-100 protein Troponin C 20-K myosin light chain (chicken gizzard) Parvalbumine Egg Albumin Chicken serum albumin Lysozome Inunoglobulin G Chymotrypsin Histone III-S

12.8

2.8 2.1

0.0 2.0

E 0:o 0.0 0.0

E 0:o 0.0 0.0

"Fluorescence measurements were performed by preparation of samples containing 10 PM of each proteins and 3 bM A.23187 in 2 ml of 50 mM Tris-HCL (pH 7.5) and 0.1 mM CaC12 or 3 mM EGTA. Each value was the mean of duplicate determinations.

some an

effect

approximate

S-100 is

on

protein,

the

binding

order

of

of

from

naphthalene-6-sulfonate

some

difference

albumin, and

III-S,

histone Ca

2+

in

15 @l

interaction

did

not

As

dialysis, fluorescence CaM

at

The

approximately

5 x 10s6

M.

CaM-dependent

enzymes,

smooth

cyclic

nucleotide

Ca2+ -dependent these

enzyme

Ca2+ -dependent

activities hydrophobic

binds

of

A23187

intensity

pH 7.5.

in

of

muscle

regions

the

to

CaM in

the

the

effect

myosin

light

up

to

203

fluorescent

by

membranes

measured

by

was

A23187

on

kinase A23187

-4 M. 1 x 10

recognized

A23187

constant

phosphodiesterase.

may

used the

calcium

two

Ca

ion

2+

and did

Therefore,

A23187

egg

chymotrypsin,

of

chain

with

G,

presence

of

the

including

to was

A23187

probes

examined,

dissociation

We examined

that

demonstrate

significantly

A23187

apparent

doses

to

increase

chain,

Z-p-toludinyl-

immunoglobulin

A23187

binding

suggest as

with

light

hydrophobic

proteins

significantly

proteins

myosin

difficult

various

lysosome,

-binding

CaM,

such

most

of Other

albumin,

-dependence.

equilibrium

increase and

serum

is

2+

results

probes

It

(17).

of

These

hydrophobic

proteins

chicken

intensity for

in

Ca

increase

(15,16).

Ca*+-binding

various

parvalbumin.

different

significant

to

fluorescence

troponin-C,

obviously

A23187

not

inhibit

the differ

from

,

Vol.

130,

No. 1, 1985

BIOCHEMICALAND

BIOPHYSICAL

Nicardipine

RESEARCH

COMMUNICATIONS

OJM)

Fig. 2. Nicardipine-induced inhibition of A23187-CaM fluorescence and Dansyi-CaM fluorescence in the presence of 50 pM calcium ion. Each CaM was used at the concentration of 15 pM. Experimental conditions were described in "Experimental Procedure". that

demonstrated

enzyme

by

activation

hydrophobic

with

dependent Figure

CaM

in

of

ICso

value

this

calcium

Ca

in

$4.

These

results

antagonist

nicardipine

that

sites

distributes

mainly

with

that

distributed free

to

binding has also monitor

to

be

with

can

not

to

cytoplasm.

in is

involved

determined.

novel

a Ca

by 2+ in

A23187

A23187 Ca

and 2+

(3),

the

3)

cell

the

fashion.

interacts

not have

regions

204

of

only proved CaM.

with

on to

cell

appeared

an and the

CaM

CaM.

membrane to

the

be

results

membrane

increased

mechanisms

the

A23187

These

the

M.

investigated

(Fig.

molecular

10 -4

in

site(s)

penetrate

with

1 x

Ca '+-binding

cytoplasm.

interacting

to

ionophore

could

A23187

-dependent

the

we

penetrates

nicardipine

-dependent

up

Ca 2t-CaM

fashion

the

A23187

in

in

ion

inhibit

binding

after

also

fluorescence

that

fluorescence

mainly calcium

cannot a dose

similar

formed

or

A23187

CaM

suggest

elements

the

in

cytoplasm

ionophore

CaM. the

in

the

calmodulin to

nicardipine

A23187

have

A23187

discrete

cytosolic

bound

CaM.

with

suggest

related

a concentration-dependent

are

whether

interact

associated

antagonists

and

activity,

suppressed

complex

these

manner,

kinase

nicardipine

fluoromicroscopically and

CaM

blocker,

-dependent

-CaM

25

and CaM

2+

2+

channel

chain

that

of

molecule

a Ca

light

2 shows

Since

calcium

myosin

presence

and

(15,16,17).

A dihydropyridine interact

probes

do and

is

concentration

of

cell

membrane

and

also

Whether

or

the

A23187

of with to

not

the

ionophore

action

the

cell

membrane

be

useful

tools

but to

Vol.

130,

No.

2i-f

3

contrast observation.

1. 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Distribution of A23187 in the cultured rabbit arterial smooth After a 5 min incubation in the presence of 1 pM A23187, phase microscopy (8) and fluorescence microscopy (A) were used for

muscle

ACKNOWLEDGMENTS We thank M. Watanabe experiments and M. Ohara manuscript.

and T. Shin for of Kyushu University

kind

cooperation for critical

with the readings

of

the

Vol. 130, No. 1, 1985

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

This work was supported in part by a Grant-In-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

REFERENCES Krestinger,

R.H. (1979) Adv. Cyclic Nucleotide Res. ll-, 1 H., and Goodman, D.B.P. (1977) Physiol. Rev. 57, 421 Klee, C.B., and Vanaman, T.C. (1982) Adv. Protein Chemistry 35, 213, Academic Press Inc. Adelstein, R.S., and Klee, C.B. (1981) J. Biol. Chem. 256, 7501 Perrie, W.T., and Perry, S.V. (1970) Biochem. J. 119, 31 Hidaka, H., Yamaki, T., Totsuka, T., and Asano, M.1979) Mol. Pharmacol. 15, 49 Lowry, O.H., Rosebrough, N.J., Fart-, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265 Hidaka, H., and Asano, T. (1976) J. Biol. Chem. 251: 7508 Hidaka, H., Yamaki, T. Naka, M., Tanaka, T., Hayashi, H., and Kobayashi, R. (1980) Mol. Pharmacol. 17, 66 Hummel, J.P., and Dreyer, W.J. (1962)Biochim. Biophys. Acta 63, 530 Blaes, N., Bourdillon, M.C., Crouzet, B., and Boissel, J.P. (1980) Cell Tissue Kinet 13, 445 Charnley-Campbell, J., Campbell, G.R., and Ross, R. (1979) Physiol. Rev. 59, 1 McClure, W.O., and Edelman, G.M. (1966) Biochemistry 2, 1908 Pfeiffer, D.R., Taylor, R.W. , and Lardy, H.A. (1978) Annals New York Academy of Sciences 307, 402 LaPorte, D.C., Wierman, B.M., and Storm, D.R. (1980) Biochemistry 19, 3814 Tanaka, T., and Hidaka, H. (1980) J. Biol. Chem. 255, 11078 Inagaki, M., Naka, M., Nozawa, Y., and Hidaka, H.7983) FEBS Lett. 151, 67-70.

:: Rasmussen, 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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