Proton NMR study of the binding of concanavalin A on myeloma plasma membranes

Vol. 104, No. 1, 1982 January 15, 1982

BIOCHEMICAL

PROTON

RESEARCH COMMUNICATIONS Pages 113-120

NMR STUDY OF THE BINDING OF CONCANAVALIN ON MYELOMA PLASMA MEMBRANES

Charles

1

AND BIOPHYSICAL

Tellier Anne

Laboratoire Facult6

’ Chantal Gbdard3

Curtet’ and Jacquks

de Chimie Organique des Sciences, 44072

Serge Aubry*

Physique, Nantes

A

1%

Poignant

CNRS

ERA France.

Cedex,

,

N”

315,

2

Laboratoire de Biologie, INSERM lJ 211, Facult6 de Mgdecine, 44035 Nantes Cedex, France. 3 Laboratoire de Biochimie, Faculte de Mgdecine, 44035 Nantes Cedex, France.

Received

September

1981

1,

The dynam i cs of proton NMK spectroscopy. of purified membranes provides a restriction Enzymatic assays and discuss the NMR results.

the

lipids

inside

Sp ec t ra collected of mobility chemical

plasma

are on

a of determinations

membranes

performed zonal rotor. the I ipids

is

investigated

using

on

are

two different fractions A Iectin, concanavalin on one of these fractions. investigated in order

A, to

INTRODUCTION All a

ccl

the

I

cells to

have

respond

plasma

to

of

(2)

and

plasma

membrane

enzymes

(3).

events

of

Various described

;k Author

early at

the

to

whom

as

and

surface.

bnt

lipid

the i I

KEY WORDS

:

-

Plasma membranes Lipid mobi I ity NMR Concanavalin A

ABBREVIATION

:

-

NMR ConA:

Nuclear Concanavalin

known

Iigands. of about

bound

of

suggested,

interacting in

with the

surface

lyrrphocytes results

be

cell

possible

been

structure of

of across

various have

cooperative

contradictory

Magnetic

the

enzymes

environment

should

Activation information

hypotheses

stimulation

now,

correspondence

:

transfer

membrane

ordered

a highly

fluidity

the

Several

such

membrane

various

is

signalling.

or

for

requires little

proteins (I),

or

I igand

transmembrane

cytoskeleton

ne

receptors

Relatively

involving

the

a

membrane.

mechanisms either

surface

by have

me&rareceptors

Con been

A

were

obtained

addressed.

Resonance A

0006-291X/82/010113-08$01.00/0 113

Copyright 0 1982 AN rights of reproduction

by Academic Press, Inc. in any form reserved.

Vol. 104. No. 1, 1982

BIOCHEMICAL

on

I ipids

the

state

A using

of

the

fluorescent We

tion

of

upon

the

and

probes

report

here

lipid

and

the

binding

of

of

in Con

membrane ESR

use

dynamics

AND BIOPHYSICAL viscosity

probes

(4,

proton

isolated

NMR

of plasma

after

the

binding

of

the

modifica-

Con

5). spectroscopy

plasma

membranes

to

study

a

murine

of

plasmocytoma

A.

MATERIALS Preparation

RESEARCH COMMUNICATIONS

AND METHODS

menbranes

The plasma membranes were purified from MFvS cells derived from the murithe ne plasmocytoma MOPC 173 grown as ascites in &lb/c mice. After washing, cells were placed in a hypotonic medium (5mM Tris HCI pH 8, 2mM CaCl2, 1mM isolated by zonal rotor centrifugation (Tl NaHC03 ) and the plasma membranes 15) as previously described (6). T wo fractions characterized by enzymatic de: F I and F II. Enzymatic assays of 5’ nucleotidase terminations were collected (EC.3.1.3.5.) and Na+/K+ATPase (EC.3.6.1.3.) were determined according to (7). For NMR studies, freshly prepared plasma membranes were extensively washed with a 20 r&I phosphate buffer pD 7.4 NaCl 100 1&4 D20. A similar concentration of membrane for fraction added directly

Chemical

proteins, I as for to the

10 mg/ml, was adjusted fraction I I. Con A dissolved NMR tube and preincubated

in for

the in 30

buffer for each sample, the same buffer was then mn at 37°C in each case.

determinations

Lipid

extracts from p I asma membranes were prepared according to the mePh osp h o I IPI ‘d s were separated by thin layer chrothod of WAYS and HANAHAN t 8). matography on plastic sheets (5 x 10 cm) with silica gel G (Met-k) using chloroform/petroleum ether/acetic acid (65/33/2, v/v/v) as solvent and after drying ch I oroform/methano I /acet ic acid/water (25/12/4/2, v/v/v/v) (8). They were revealed by exposure to iodine vapor and the amount of inorganic phosphate of the different spots was determined according to (7). Cholesterol was estimated with a Biochemica Test Combination Kit (Boehringer, Mannheim). The fatty acid composition was determined by gas chromatography (Carlo Erba) after methylation with a capillary column filled with Carbowax 20 M. The standard fatty acids were obtained from Alltech Ass.

NM3 spectra Proton NMR spectrometer obtained from experiment.

NMR

experiments operating 1000-2000

were 250

at transients

performed on a CAMECA MHz. Accumulated free at 37°C corresponding

250 Fourier induction to 20-45

Transform decays were mn for each

RESULTS The of

NMR

material fractions

rences

in

tions described

analysis

purified

brane

pid

spectrum

classes (data

by termed

the

zonal F

I

lipid/protein (Table not

previously

II).

The 0th

shown).

of

plasma

rotor and

F

required

centrifugation. I I

were

ratio

(Table

fatty

acid

er

membranes

Two

obtained I) profile

characteristics

and

and the was of

(6).

114

a purified

severa

distribution

these

amount

plasma

displayed

equivalent

large

of in membranes

memI

di ffe-

phospholi-

both

populahave

been

Vol.

104,

No.

BIOCHEMICAL

1, 1982

AND BIOPHYSICAL TABLE

Chemical and

conposition fraction

I

of plasma membranes (Results from four

II

RESEARCH COMMUNICATIONS

from fraction preparations).

F Prote

in

Total

lipid

Lipid/Protein

(W/W)

Phospholipid/Protein

(W/W)

Cholesterol/Protein Ch 0 I es terol/Phosphol (molar ratio)

The shows

proton

NMR

several

CH2-CO-,

higher

in

fraction

II

in

fraction

I

constituents

of

spectra, (cf.

Table wel

fraction

of

trum

of

sity

and

I .

the a

line

The

data

proton

Con

the

A

NMR (up

to

revea

67.9

49.4

2.12

0.98

0.74 0.13 0.37

0.11

I

0.60 0.38

and

terminal latter

I

I I

(f iguresl

CH3-,

I ipid

signal

was

of

which

that

fraction

broadening

of

the

was

the

and

2A)

chain

-CH2-,

on I y

seen

higher

most

(figure

resonance

TABLE

fraction

composition II

Phospholipids

of (Results

obtained

ratio

A greatly II

CH2

spectrunwas the

Con

contrary,

membrane

Phospholipid and

50.6

ion

concentrat

These

the

of

On

plasma

protons.

relative II).

to

II

32.1

membranes due

F

I

than

mobi

I ipid

le

I resolved.

amounts

with

)

33

the

modification

increasing

plasma peaks

+ -N-(CH

and

are

of

resolution

-CH2-CH=C

No

spectrum

1:;’

I

Con

the Both

peak

were

a

proton

(%)

membrane from four preparations).

decrease

in

produced.

fraction

I

F I

F II

29.3

24.5

15.1

15.0

Phosphatidylcholine

27.9 26.5 1.3

37.6 19.7 3.1

ethanolamine

Sphingomyalin Lysophosphatidylcholine

115

1: 1) NMR

I I

plasma from

binding

protein

Phosphatidylserine

Phosphatidyl

the

A/membrane

modified 28).

upon

specinten-

Vol. 104, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I -CH2-

IOOHz Sucrose A

FIG.

1

The and

was

expressed

-CH

-

sample

way

in

the

in

order

in

lipid

amount

of is

to

exclude

to

membrane

dynamics from

Con

the

A

given

of

I of plasma n&4 pD 7.40,

the

(figure

the

ratio 3).

of This

possibility

of

agglutination

and

Con

A was

value

l/10

- l/4

raise

the

nature

only.

mobility

of Fraction buffer 20

37°C).

the

due

I ipid

MHz spectra a phosphate

intensities

inhomogeneity

decrease

brane

to

peak

this

differences

portant

respect

resonance

3

in

portions of the 250 from MF S ce I Is in d, D20. t 1000 scans,

with

in

increase

proved

to

ratio

any to

smal

Con

I

account

cause

mg

the

an A/mg

immem-

proteins. The

between lipid

multiple and

extract

the

binding

receptors of

vesicles on

The upfield membranes N&l 100

variation

-CH2-

for

-

was proton

proteins

in

sites

I i p i ds.

plasma to

NMR

spectrum

those

A

vesicles

The

resu

obtained was

seem

Con

Membrane

membrane.

similar

particular

of

were It ing

in

monitored.

necessary

F The

to

and Con

induce

interaction

reconstituted

proton I

of

NMR F

il.

No

A receptors,

with spectrum

of

these

effect

of

Con

ng

glyco-

hi ndi

a perturbation

total

of

lipid

A

dyna-

mics. We binding

also of

checked Con

A.

the Significant

activity

of

the

inhibition

membrane of

116

enzymatic

bound

5’ specific

nucieotidase activity

upon was

Vol.

104,

No.

BIOCHEMICAL

1, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-CH2-

Sucrose

n 100 Hz

Sucrose

,100 Hz

FIG.

2

observed It

-

upf i e Id port ions of the 250 MHZ proton NMR spectra of fract ion of plasma membranes from MF2 S ccl Is in a phosphate buffer2OnM, 7.40, N&l 100 n+i, D20 (2000 scans, 37”Cj.A : without Con A. : after incubation with Con A (0.25 mg Con A/mg membrane proteins). : signal from Con A.

with

reached

NMR

The I I pD 6 X

increasing a

spectrum

absence

of

amounts

p I ateau of

Con

at

plasma

SO-60

of

Con

Th e

%.

membrane

A with absence

of I

fraction

the a

does

two

fractions

Con

A effect

not

seem

membrane

of

(figure on to

the

depend

4). proton on

the

A receptors.

DISCUSSION The toma the

was membranes

effect

of

monitored

Con by

with

A binding proton

Concanavalin

to

the

NMR.

This

A

reduced

117

plasma technique the

a

revealed mobility

murine that

of

the

plasmocytreatment

chain

-CH2

of pro-

Vol. 104, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

I I I ‘~100

If200

I 11

‘/so Con

3

FIG.

tons

from

-

Ratio the tion

the

A

lipids.

I inewidth

has

the

contribution

major

of intensity ratio of Con (fraction II

been

A/

‘ho

‘I4

?I2

membrane

prorein

(W/W)

of methyiene peak over methyl A concentration over membrane of plasma menbranes).

quantitative

attempted to

RESEARCH COMMUNICATIONS

interpretation

by the

several resonance

peak proteins

of

(9).

authors linewidths

> plotted against concentra-

the It

of

-C%-

has

been

resonance shown

phospholipid

bilayers

R&live specilic activity t

I o9

’ l/200

‘400

I

I

“50

‘40 ConAhmbrone

FIG.

4

-

I ‘4

Modulation of the 5’-nucleotidase activity of Con A to fraction I (X . . . . . X) and relative specific activity is the ratio vity in the presence of Con A and the the lectin.

118

protein

1 ‘12

1

1

‘4

(W/W)

by adding various amounts fraction II (0 . . . . . 0). The between the specific actispecific activity without

that

Vol. 104, No. 1, 1982 is

dipolar

in

methylene to

of

other.

low

the

the

membrane

resonance

some

rement

of

cross

use

tional the

lipid

The

reason

were

in

ler I

membranes rent

of

I I

chain

to

averaged as

by

the

large

rapid

ampI

itude

broadening

excludes

the

of

of

vesicle

tum-

restricted

mobility

I when

the

with

the

bi

remained inside

of

to

fluidity

out

13).

obtained

proton

NMR

use

of due

without

enzyme

of

measu-

the

any

restricted

of

ConA

discrepancies

produces

responses

the The

by

shows

correlate of

(12,

layer

modulation

between

of

order

complexes

binding

addi-

mobility

activities.

fractions

I and

II

upor

determined. examination

size

Is

use

A

ion

in

probes

the

the

difference be

ccl

resonance

Con

proport

tried

lipid

By

but

A. been

the

diminished

the

receptor

14). that

was

and

than

rather in

large

fraction

pellet

was

plasmocytoma

of

cells

I I .

the

vesicles

(0.1,

to

The

suspended were

authors in

broken

1,)

a

up

(6)

showed

but

with

a mean

deduced sucrose

into

that

that

size during

solution, fragments

they

plasma with

diffe-

properties. Another

and

in

the

lectin

spin

microscopic

or

acid

such

zones

Con

and

correlates

to

fraction

lysis,

by

of

shown

the

heterogeneous

smal

the

(5,

which

remains electron

F

of

have

electon

have

for

A binding An

of

probes

bilayer

account

membranes

and

we

fatty

differential

groups

affected

properties

these

probe,

The

A produces

approaches

dynamic

of

diffusion,

the

submitted

partially

lateral

methylene t ake

plasmic

probes

the

the

motions

of

Con

the

not

linking

the

fluorescent

Con

were

of

by

of are

spectrum

that

could

several

now,

properties

receptor

of

This which

Until

end

(11).

NMR

of

peak

lipids

resonances

molecular by

because

lipids.

sharp.

vesicle

intra

rotation

seems

individual

one

phospholipids

proton it

plasma

relatively

to

the

difficult

interactions

and

vesicle

contribution,

The

ccl

and in

from

inter of

I inewidths

of

dipolar

frequency

RESEARCH COMMUNICATIONS

remains

superposition

magnetic

motion

ing

a

interpretation

proceeding

reorientation

segmental

AND BIOPHYSICAL

but

motion

However,

angular

in

(10) is

gradient

relatively

bl

origin

resonance

a

the

BIOCHEMICAL

right

interesting side

out

data vesicles

might in

the

be two

the fractions,

119

relative

proportion which

might

of change

inside the

out degree

Vol.

104,

of

No.

Con

A

fifty

of

in complete

lectin

was

each

form

is

absence

and

the

proton

fraction

Con

A

ratio ratio

;

linking

in

the

of

Con

that

but

the

as

shown

is

is

right be

from

enzymatic

probably

I ipids

A receptor

the

I ized too

low

fraction

I

side

out

explained

by assays.

related

i&i

probably

about in

cannot

before I

of

regularly form

of

results

fraction

amount

was

inside-out

proportion NMR

COMMUNICATIONS

there

more

and

receptors

RESEARCH

(15) I I

negligible

protein-lipid

cross

shown

in

not

of

receptor-lipid

ring

BIOPHYSICAL

Nethertheless

I

low

AND

previously

percent).

fraction

The

by

It

seventy

form

BIOCHEMICAL

binding.

percent

(about

a

1, 1982

to

a

by

the

cluste-

to

be

detected

low

NMR.

ACKNOWLEDGMENT The

authors

thank

Professor

M.L.

MARTIN

for

helpful

comments.

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