In vitro interaction of 1-anilino 8-naphtalene sulfonate with excitable membranes isolated from the electric organ of Electrophorus electricus

Vol. 36, No. 3, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IN VITRO INTERACTION

OF l-ANILINO

SULFONATE WITH EXCITABLE

8-NAPHTALENE

MEMBRANES ISOLATED

FROM THE ELECTRIC ORGAN OF ELECTROPHORUS ELECTRICUS

Michiki de Biologie

Departement

RASAI, Jean-Pierre CHANGEUX MolCculaire, Institut Pasteur,

Paris

and Lucien MONNERIE Laboratoire de Chimie Macromoldculaire, Ecole de Physique et Chimie, Paris

Received June 16, 1969 Summary- In vitro binding (ANS) to membrane fragments derived of electroplax cells is accompanied intensity and polarization. Effects that a structural reorganization of 30 and 5O'C. Fluorescence polarization of solvent viscosity upon addition difies ANS binding. I. 1-anilino

present

probe

fragments

(EME).

It

strong

enhancement

is

is

we describe derived

shown that

interpreted

effect

bound

to EME indicates

of the isolated

membrane

trast

is

what

viscosity

structure

the

interaction

the

generally

in vitro

innervated

face

intensity

membrane

on the fluorescence

occurs,

observed

upon addition

with

used In the

of ANS with cells

accompanied

by a which

hydrophobic parameters

structural

areas. of ANS

reorganization

between

30 and 50'12.

soluble

proteins,

of sucrose

420

(l-10).

and polarization,

an irreversible

fragments

comnly

of electroplax

of ANS to EME is

of ANS with

of temperature that

(ANS) has been

and membrane

the binding

as an interaction

of the

of solvent

from

of fluorescence

Study

with

sulfonate

of protein

communication

membrane

INTRODUCTION

8-naphtalene

as a fluorescent

of 1-anilino 8-naphtalene sulfonate from the innervated, excitable, surface by a strong enhancement of fluorescence of temperature on ANS binding indicate the membrane fragments occurs between of bound ANS is insensitive to changes of sucrose. Ca++ and d-tubocurarine mo-

is not

In con-

the change

accompanied

Vol. 36, No. 3, 1969

BIOCHEMICAL

by any significant This

last

alteration

effect

a membrane

appears

structure.

in vitro

of fluorescence

polarization

to be a characteristic

property

Several

of the electroplax,

EME were

following (11).

the method

ding

ANS.

of ANS bound

of the excitable and flaxedil,

to

membrane

modify

the

by differential described

all

tissue

in sucrose

Gautheron,

Israsl

the experiments

and Neel

were

of ANS were

(12)

with

from

gradient

and Podleski

and 5x10m3M glycyl-glycine and polarization

of Monnerie

electric

centrifugation

indicated,

intensity

to the method

of fresh

by Changeux,

of 0.2 M sucrose

Fluorescence

AND METHODS

from homogenates

when otherwise

in the presence pH 7.0.

MATERIAL

prepared

electricus

Except

d-tubocurarine

of bound

of ANS to EME.

II.

Electrophorus

effecters

in vivo

Ca++ ions,

interaction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

performed

buffered

at

measured

accor-

a fluorescence

polarization

apparatus. The wavelength observed

at 438 mu ; in both

polarization IIh

of excitation

being

directions

p is the

defined

intensity

with

instances

exciting

enhancement

and horizontal

light.

of EME to ANS in 0.2 M sucrose

similar

serum albumin

(BSA),

change

absorption

difference

vertical

RESULTS AND DISCUSSION

of ANS fluorescence.

to EME is

observed

the

of AN.5 to EME Exposure

bound

filters were used. The Vv- IIh p = ITv+ Ish , *Iv and n

p = - 2pn where Pn' 1 components measured along

unpolarized

was

interference

as

III. 1. Binding

was 365 mu and fluorescence

of the with

The fluorescence

in shape and its

spectrum

between

to the spectrum

maximum occurs

BSA : the absorption

spectrum

is

bound

accompanied

emission

at 365 mu shifts

and free

421

is

ANS also

shows

similar

of ANS

to bovine

same wave-length.

of ANS upon binding band

spectrum

of ANS bound

at the

by a strong

The to the one

to 371 mu, and the a maximum at 390 mu.

Vol. 36, No. 3, 1969

BIOCHEMICAL

The amount complex

ANS-BSA

intensity

(If)

of ANS bound

ANS in the

protein

per

was found

centration complex

similar

with that

result

mole

lo5

per

g of protein

(n)

methods

the difference

yield

in optical

; 3) various

and the

concentration

of

by optical

density

measurement

of sites

per mass of membrane

by extrapolation

same as for

and the the complex

at infinite

extinction ANS-BSA

ANS con-

coefficient i.e.

0.75,

of and

at 390 mn (Fig.1).

of ANS bound

In the present

speed

was determined

indicates

ronment.

at high

the quantum

concentrations

ANS was determined

The same number

the

range,

to the

of fluorescence

of increasing

a smaller

and free

the three

per mole

to that

bound

solution

ANS-EME are

This

within

by reference

: 1) the variation

as a function

centrifuged

mole).

assuming

AOD = 2.04~10~

is

were

supernatant

(E350 = 5x10'

the

but

at 390 mu between

ANS-EME mixtures

manner

measured

of ANS ; 2) in parallel, density

to EME was estimated

in the following was first

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

the

environment

to BSA, namely

experimental

conditions

a highly

hydrophobic

we determined

= 4.4x10m4M.

and ED = +

of ANS in the membrane

Of interest

envi-

: n = 6 +-1 is

the

A

. fluunscence lntenstty o difference spectrum x ultracentrifugation

1

1

Il)x 104

2Dx104

M-’

freek Figure 1. - Quantitative estimate of ANS bound to EWE. Three different kinds of experiments were carried simultaneously 1) the fluorescence intensity of bound ANS was first measured, then 2) the difference in optical density mixtures of ANS and ESE at 390 mn between free and bound ANS ; 3) various were centrifuged at 55.000 EPM in a SW 65 swinging bucket rotor for 90 minutes and the optical density of the supernatant was determined. The concentration of membrane was 1 mg/ml in proteins (using the Folin reagent and BSA as the standard). The temperature was 22'C. 422

BIOCHEMICAL

Vol. 36, No. 3, 1969

fact

that

the

the variation

reciprocal

large

of the

of free

range

with

at 20°C a value

same conditions. EME as it not

interact

2. Effect

If

and p for

is

progressively with

the solvent

to their

affinity

almost

light

identical

changes

as much as they

Pig.2

of l/p

respect

of the

with

suggests

follows

that

of pure

within

a

sites

ANS.

with

to EME

BSA under

of ANS molecules

ANS binding

sites

the bound

on EME do

on ANS binding.

to EMS in 0.2

elevated T/n

found

with

do on BSA.

and 3 show the variation

ANS bound

for

by ANS bound

the number that

line

the ANS binding

emitted to that

of ANS bound

a straight

that

BSA which

of temperature

follows

indicates

(p)

p does not

does with

of the number

which

of concentration,

The polarization 0.31

reciprocal

ANS concentrations

on EME are homogeneous

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

M sucrose.

0 to 3O"C,

the Perrin sucrose

as a function

If

When the

for

of

temperature

and p decrease.

law assuming solution.

of temperature

The variation

the viscosity

The rotational

(T)

relaxation

(n)

of time

\ 0.2

0

.

\ x 01 * ’ . ’ ’ ’ s 0 30 60 Temperature ‘C

’ ’ ’ . ’ 30 60

’

0

Figure 2. - Effect of temperature on ANS binding to ESE. The concentration of ESE was 0.2 me. of nroteins ner ml and that of ANS 2x10e5M. The experiment was carried in the presence of-O.2 M sucrose and 5x10-3M glycyl-glycine-HCl pH 7.0 at 20°C. Control experiments with BSA were done in the presence of Readings were made 5 minutes 2xlO-7M ANS and 0.2 mg/ml of BSA in buffer. after temperature equilibrium. 423

to

Vol. 36, No. 3, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T/,,xIO-~ 0 I VP

I

2

3

4 0/r 4ooc

x-x

Tconetent.

Figure 3. - Compared effects of sucrose on the polarization of fluorescence of ANS bound to ESE and BSA. The concentration of ESE was 0.2 mg/ml in proteins and that of ANS 2.2~10~~. When the temperature was varied the sucrose concentration was 0.2 M. When sucrose concentration was changed the temperature was maintained constant at 22'C. In the case of BSA when the temperature was varied the solution was made in buffer only. The concentration of ANS was 1.1x10-7 M and there was 0.2 mgfml of BSA. The buffer was 5x10-3M glycyl-glycine pH 7.0. p calculated life

time

close

from

found

A plot the

of binding

data

of the excited

to that

perature,

these

is

state

with

again

of log KA against

slope -4.9

more slowly to decrease

0 and 3O'C. sively

but

and p with meters

If

of ANS r = 15.5

corresponds KCal

l/T

than

linear

and with

a slope

ANS JNE is

T now strikingly

differs heating

within

an entropy

O0 and 3O'C.

to O'C,

before

n seconds.

in the temperature

between

the mixture

is

yielding

cooled

recorded

assuming

for

Such a value

this

to AH0 = -5 KCal per mole

per mole,

rapidly

rapidly

at 22'C

the is

BSA.

Above 30°C a break crease

is p = 156 n seconds

it

first is

from

to 80°C. 424

range

of tem-

at 20°C,

the AF"

contribution

curves

occurs,

Above 50°C,

If

close

to that

heated

observed

If

and p de-

and p start

measured

between

at 80°C and then that

the variation The curve

of -0.3

the variation of the

does not

progresof If

same para-

show any discon-

e.u.

Vol. 36, No. 3, 1969

tinuity

BIOCHEMICAL

and the plot

thermodynamic

parameters

AH0 = -1.64 e.u.

of log

KCal

p is now 380 n sec.

binding

sites

pretation

the size were

and shape

noticed

average

ture

is

that

and is

although

does not

interpreted

curve

are

to BO'C.

The

following

:

the

KCal per

mole,

of both

ANS is

AS' = -12.5

the number

observed.

By light

particles.

of the particles

30°C thus

this

from 0'

structural

we followed

No detectable

changes

above 55°C an irreversible

correspond

as a rearrangement

of structure of the

transition

alteration

of membrane

in parallel

increase

The structural

to a gross

inter-

reorganization

scattering

was noticed.

of ANS

The simplest

an irreversible

30 and 5O'C.

55'C

linear

an increase for

of membrane

below

size

around

results

RESEARCH COMMUNICATIONS

T constant.

affinity

between

is

AF' at 2O'C = -5.29

treatment

and their

of EME occurs

l/T from

assuming

heat

of these

K agains

calculated

per mole,

Following

AND BlOf’HYSlCAL

occuring

of membrane components

struc-

within

the membrane. 3. Effect. It tested

has been

in this

to these

shown with

respect

proteins

that

the polarization

increases

the concentration

the few globular

with

of sucrose

at any point

of the heating

and cooling

fluorescence

with

bulk

of soluble

in the organ

course

respect

was it

purified

from

Columbia

University),

purified

electric

observed organ

E. coli

membranes

total

supplied

in all

these

parameters (kindly

from

the protein

(kindly

although

of ANS fluorescence

with

the

supplied

425

from 0.2

this

was not observed

separated from

Moreover

3 when

is

homogenate

of ANS

with

the membrane

M

true

Such an independance

viscosity

were

of EME purification

; neither

dependance

which

of ANS bound

of EME is changed

curve.

have been

As seen on Fig.

of p occurs.

to solvent

proteins

viscosity.

in a suspension variation

which

of fluorescence

solvent

up to 1.5 M, no significant

proteins

the

fragments

of electric

acetylcholinesterase by Dr.

last

W. Leusinger

instances

was noticed. by Dr.A.

a temperature

On the other Siccardi

from

from

hand the Institut

Vol. 36, No. 3, 1969

Pasteur)

show the

viscosity brane

BIOCHEMICAL

sems

phenomenon thus

; the independance

to be a characteristic

of p upon increase

property

of solvent

of ANS bound

to a mem-

structure. In addition

fluorescence tration

we noticed

emission

of sucrose

emission

of ANS bound

of ANS bound

upon

addition

strongly

2) since viscosity,

from 0.2

direct

Various tested

nity

of ANS to its

versene.

Carbamylcholine

no significant

change.

effect

d-tubocurarine

towards

concenthe

shorter

sites

increase

The effect

or flaxedil, respectively

of the number

of ANS binding

whether

these

effects

action

of Ca

are

sites. related

of these

it

binds,

the solvent

phase. results

It

should

requires

to ESE. membrane

of the eel

by a factor is

of 5 but

reversed

have of

the affinity

trying

in vivo

of

in the presence

of 2 and 1.6 without

We are presently to the

activators

inhibitors,

by a factor

the number

by addition

two receptor

two receptor

electroplax

of 10D3M Ca++ the affi-

up to 10 -4 M but

or not

in

the membrane

of Ca’+

at concentrations

of p measured,

to which

by changes

In the presence

and decamethonium,

of ANS to EMB increases

++

within

ANS bound

ANS-EME.

membrane

does not

when the

values

component

of the excitable

on the complex

of sites

the high

interpretation

of T for

effecters

were

for

be perturbed

a definitive

measurement

changed

a 2 mu shift

by the membrane

of ANS cannot

that

of the maximum of

M up to 1.5 M ; in contrast

to BSA showed

ANS seems to be sequestred

be emphasized

51clO-~M

to EME was not

1) accounting

immobilized

the motion

the wavelength

of 1.5 M sucrose.

Thus we see that ANS is

that

was increased

spectrum

wavelengths

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

change

to see

pharmacological

and curare.

This work benefited from the aid of the “D&lEgation G&r&ale at la Recherche Scientifique et Technique (convention no 6y-Ol-Ogl)“, of the National Institutes of Health, of the “Centre National de la Recherche Scientifique”, and of the ‘%ommissariat 1 1’Energie Atomique”.

426

Vol. 36, No. 3, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Weber, G., Adv. in Prot. Chem. 8 (1953) 415. Anderson, S.R. and G. Weber, Biochem. S (1969) 361. 13 (1965) 482. Stryer, L., J. Mol. Biol. Stryer, L., Science 162 (lz8) 526. McClure, W.O. and G.cEdelman, Biochem. 5 (1966) 1908. Gally, J.A. and G.M. Edelman, Biochim. Bizphys. Acta 94 (1965) 175. Tasaki, I., A. Watanabe, R. Sandlin, L. Carnay, Proc.?at. Acad. SC. US 61 (1968) 883. ChanceFB., A. Azzi, L. Mela, G. Radda and H. Vainio,FEBS Letters 2 (1969) 10. Vanderkosi, J. and A. Martonosi, Biophys. J. 9 (1969) A235. Freedman R. and G. Radda, FEBS Letters 2 (1967) 150. Changeux, J.P., Gautheron, J., Isragl, M. and Podleski T., C.R. Acad. SC. Paris (1969) in press. Monnerie, L. and J. Neel, J. Chim. Phys. 61 (1969) 504.