BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

INHIBITION OF Cl- BINDING TO ANION TRANSPORT PROTEIN OF THE RED BLOOD CELL BY DIDS c 4'-DIISOTHIOCYANO-Z,Z'-GTILBENE DISULFONIC ACID) MEASURED BY r3 StClINMR Y. Shami,

J.

Carver,

S. Ship

and A. Rothstein

Research Institute, The Hospital the Department of Cell Biology, (J.C.), Toronto, Ontario Received

March

for Sick University

Children, and of Toronto

22,1977

SUMMARY: The width (at half height) of the NMR spectral peak of [35Cll was increased significantly in the presence of either red blood cell ghosts or a Triton X-100 extract of the ghosts, indicating chloride binding in both preparations. The specific widening was a linear function of protein concentration of the medium, amounting to 4.2 Hz/mg of ghost protein and6Hz/mg of Triton X-100 extracted protein. If cells were treated with the irreversible anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonic acid), prior to the preparation of the ghosts, the specifiti widening of the NMR peak was diminished by 32% for ghost protein and 50% for the Triton X-100 extract. The effect of the inhibitor on the widening waslargely limited to the pH range 6.5-8.5. The data suggest that the effectiveness of DIDS as an inhibitor may be related to its capacity to displace Cl from anion binding sites in band 3. INTRODUCTION: with

an intrinsic

usually data

Anion

called

location

that

site

be overcome

measured

variety

which (13),

is

of Clenriched

large

we used the

specific

to distinguish

the

in band

number

Copyright 0 1977 by Academic Press, Inc. All rights of reproducrion in any form reserved.

ghosts,

3 protein

sites

which

to a Triton red

can,

of anion might

sites

are

in proteins we have

X-100 cell

sites

extract

from

sialoglyco-

for

permability,

be involved

however,

times

and phospholipids. binding

by

of the

technique,

and the

of non specific inhibitor

trans-

affinity

binding

kinetic

proteins

relaxation

this

(1,2,3,4,5)

its

The difficulty

Using

sialoglycoprotein

irreversible particular

cell

apparent

the chloride

(10,11,12).

to red

precedes

anion

associated

Although

to membrane

the

of even low affinity

is

of 95,000

sites

67 mM) (8,9). because

and to purified

of the potentially

because

cell

et a1.)(6).

of such binding

low(Km=

[35CllNMR,

weight

to membrane

feasible

of sources

the binding

the ghosts proteins

is

by the presence

from a wide

binding

is not Cl-

by using

molecular of Fairbanks

a demonstration

for

the human red blood

3 (nomenclature

methods

transport

in

of apparent

chloride

(7,8,9),

conventional

affected

protein band

suggest

permeability

in Cl-

Because chloride, (DIDS)(l,Z) transport.

429 ISSN

0006-291X

Vol. 76, No. 2, 1977

Figure

1.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

The line width at half height of t35Cll resonance in (A) 250 mM KCl, (B) 250 mM KC1 plus Triton X-100 extract of red blood cell ghosts (5 mg protein per ml), (C) 250 mM KC1 plus Triton X-100 extract of DIDS (10 pM) treated red blood cell ghosts (5 mg protein per ml).

MATERIALS AND METHODS: Human red blood cell ghosts were prepared by the method of Dodge et a1.(14) and were extracted with 1% Triton X-100 (Rohm and Haas, Canada)-at ionic strength, 40 mM by the procedure of Yu et al.(U). The fraction extracted by this method is enriched in band 3 and the major glycoThe extract was then concentrated proteins and also contains phospholipids. eight to ten fold with a Diaflo XMlOOA filter to final concentration of 5.0 to 10 mg protein/ml under 15 psi N2, the chloride concentration was adjusted by the addition of KCl, and pH was adjusted by the addition of either HCl of NaOH. To evaluate the Cl--binding of major constituents other than band 3, the Triton extract was passed through an organomercurial sepharose column (15). Some of the sialoglycoproteins and all of the lipid components in the extract were eluted from the column but all of the band 3 wags retained (15). The eluate was tested for its broadening effect on the [ CllNMR line width. In addition, the glycoprotein components were separately purified (16) andtested. Except in the experiment of Fig. 3, the pH was 7.2. The treatment of red blood cells with 10nM DIDS (4,4-isothiocyano-2,2'-stilbene disulfonic acid) was carried out as described previously (2). Protein was determined by the method of Lowry et a1.(17) and sialic acid by the method of Aminoff(18). Samples were kept at 4*C except during NMR readings which were carried out at 23oS. Pulsed Fourier transform NMR spectra of chloride were obtained on a Bruker BKR-322 variable frequency pulsed spectrometer. For a field equivalent to proton resonance at 60 MHz, [35Cll resonated at 5.88 MHz. Chloride NMR line widths were measured at half height of the spectral peak. RESULTS: half

Typical

height

as a blank T&ton

X-100

for for

[35C11NMR spectra

are

shown

250 mM KC1 was 19 Hzk0.G the

extracts

calculation

of line

S.E.(Fig.

width

of human red blood

430

cell

1. The line

in Fig.

lA),

broadening. ghosts

a value

width that

at was used

In the presence (5 mg protein

per ml),

of

Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

mg pr%ein

per ml

10

CONTROL

60-

50-

DIDS

40N 30=2D‘O-

A

o+

,

,

,

,

, 5

0

the

line

2.

width

was considerably

extracts

ml),the

broadening

4.2

from

cells

treated

as well

as Triton

was linearly

the

cells

decreased

in each case.

was 50% from

6.0

of the

were

(Fig.

increased

at the p CO.01

width

proteins

to the amount

with

In the ghosts, 2A),

and in

lines

with

DIDS,

the Triton

431

was only

X-100

extract,

to

extracted widening 4.2

the

The difference

DIDS in both

case the

amounting

was 32%, from

2B).

cell

In either

added,

X-100

(Fig.

36Hz(Fig.lC).

of red

the specific

the decrease

level.

5 mg proteinper

(Fig.2).

of protein

and without

but with

presence

and 6.0 Hz per mg of Triton pretreated

lB),

containing

in the

to 3.1 Hz per mg of protein

regression

(also

The line

extracted

protein

If

Hz per mg of protein

X-100

DIDS

less.

width

related

protein.

significant

, 10

to 52 Hz (Fig.

with

was substantially

Hz per mg of ghost

slopes

,

per ml

broadened,

The [ 35C11NMK line

increase

,

Variation in [35C1] (250 mM KCl) Nick line width as a function of protein concentration. (A) Ghosts (e) control; (0) from DIDS treated cells, (B) Triton X-100 extract of red blood cell ghosts (e) control; (0) from DIDS treated cells.

similar

ghosts,

,

protein

mg

Figure

,

cases

to 2.7

decrease in

is

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PH Figure

3.

The excess L3'C11 (250 mM KCl) NMR line width as a function of pH. (0) ghost protein; (0) Triton X-100 extract of red blood cell ghosts; (x) extract from DIDS treated cells. The protein concentration was 5 mglml in all cases.

The line was maximal

broadening

at pH 7.2

broadening

for

(Fig.3).

Except

per mg of protein

extracts

than

pH because

for

the

the ghosts

ghosts. had almost

cells,

of DIDS at low or high to pH values

in no irreversible

higher

ranging

effectsinterms

the for

extracts

specific

the Triton

X-100

large

on the NMK spectra

at low

below

pH 6.

at pH 7.2 was eliminated,

of pH were from

X-100

was especially

the peak values

or Triton

pH values,

no influence

the effects

resulted

at high

The differential

from DIDS-treated

or extracts

the ghosts

was substantially

In extracts

ghosts

either

minimal.

3 to 10 for of their

Cl-

but

Exposure

to

up to 3 hours binding

measured

by

NMR. To assess X-100

extracts

carried measured albumin).

out.

other Triton

the possible than X-100

band

effects 3 on the

(1%) itself

at pH 7.2 in the presence The extract

contains

of major line

width,

had no effect

or absence phospholipids

432

components several on [

of protein

in

the Triton

controls 35

CllNMR

(Bovine

were spectra

serum

(20 Vg per mg of protein)

and

BIOCHEMICAL

Vol. 76, No. 2, 1977

protein 15%)

components, (4,13).

largelyband

An eluate

an organic

mercury

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3 (about

80%) and sialoglycoproteins

of the Triton-extract

column

(prepared

- see methods)

containing

(about

by passage only

through

the phospholipids 35

and some sialoglycoproteins NMR line result

width

(values

was obtained to those

fore,

3 is

band

bute

largely

is

the only

across

the widening generally

protein

X-100

of Cl-

to protein

ratio

protein

quantities

but

(about

widening

of the [

cluded

as contributors

effect

is

CllNMR

the broadening

extract

signal,

of evidence

in

do not

contribute

433

is

of the band

was carried

out with

Such extracts in about

are

the

about

are present

30% Apart

in only

the phospholipids significantly

components

This

3

in the extraot.

that

to the relative

protein

Because

Band 3 constitutes

proteins

3.

directly, of ligand

purified

study

seems likely

to band

binding

phospholipids

minor

it

addition

per mg of ghost

(10).

indicates

Although

of Cl-

Cl-

3 protein.

(13).

study

its

based

in the presence

to ligand

in band

to the widening,

due to the binding kinds

signal

other

to precede

indirect,

to measure

80% of the protein

The present in the

35

is

the present

15X),

(amounting

Cl-against inhibitors

and contain

about

contri-

contribute.

of undenatured,

enriched

there-

could

has been assumed

as the ghosts

(5),

(4,13).

and sialoglycoproteins

by several

amounts

NMR measurements,

the sialoglycoproteins

small

(I),

as binding

of ghosts

also

the evidence

absorption

Cl1 The same

components

oronprotectionby

in sialoglycoproteins

ghost

might

[35C1]NMR

level).

that

Minor

an opportunity

the large

extracts

enriched

same lipid

from

of the

but

[

By elimination,

the extract

3 protein

(7,8,9)

offers

for

in

control

the

in concentrations

extracts.

component

to band

increase the

[35C11NMR signal.

behavior

necessary

significantly

the Triton

the membrane,

of preparing

not

in the extract)

interpreted

difficulty

of the

in major

The NMR technique

also

sialoglycoproteins

of Cl-

on kinetic

Triton

purified

of the

Binding

translocation

for

1 to 2 Hz above

5% of the protein

DISCUSSION:

(19).

only

present

to the broadening

to about

did

were

using

equivalent

(15)

that

to the

cannot

be ex-

most of the

conclusion

is

abundance

of the same order

supported of band of magni-

3:

BIOCHEMICAL

Vol. 76, No. 2, 1977

tude

as that

a minor

reported

component,

large;(II), verse

the excess

the exchange times, ated

field

and on this for

the bound

rotational weight

is most

likely

using

correlation

time

for

the quadrupolar

(12);(III),DIDS

is

found

highly only

in phospholipids

(2,21,22).

Its

conclusion

that

supports

band

the

trace

width

the Triton

X-100

inhibitory

effect

If band

3 contributes

protein

and almost

could

account

binding

(Fig.

Cl-

amounts

(20),

constant.

binding

in band

Using

distributed

a value

90%) with

inhibition

DIDS-modulated

of

value

metal

in other

a

molecular

This

to other

3 (over

substantial

can be estim-

is

free about

in

proteins 5% in the

proteins

and none

of the NMR peak widen-

Cl-

binding

is

located

is

somewhat

in

3. The line

the

for

localized

sialoglycoprotein,

ing

coupling

and

to the relaxation

from an estimated micelle

of this

band 3 extract,

et a1.(12).

obtained

values

X-100

of Nome

X-100

for

complex

the procedure

3 Triton

trans-

process

ion-protein

to contribute

derived

of the

Modulation

constant

the band

with

site.

coupling

of 16 nsec

due to

to be unusually

relaxation

a quadrupolar

for

good agreement

have

measure

For Triton

slow

largely

of the quadrupolemoment

of the

ions.

were

a direct

binding

rotation

too

of 310,000

2 MHz is

it would

the interaction

the

and free

ion

If

The dominant

at the ion

bound

assumption

is

Cl-.

from

from both

process

width

gradients

between

(11,12).

per mg of protein

[35Cl]line

arises

can arise

exchange

proteins

of the bound

nuclei

the electric

from

of

time

such quadrupolar

interaction

other

the broadening

relaxation

with

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

must

line 3).

accessible

broadening

extract

for

of DIDS is

Additional

mg of protein

in the ghost also

higher

all the

is

(4.2 in

a disproportionate

accompany

broadening

than

per

the extract

fraction

Triton very Cl-

as a consequence

In addition, X-100 low in

binding

its

however,

extraction. but

ghosts, sites

enrichment

in

in

and the

by ghost the extract

this

in Cl-

at pH 5.5

high pH range

in

in

to 50%).

some alteration

relatively

extraction.

434

Hz/mg)

(35% compared

For example,

visible

of detergent

to 6.0

of the broadening

then

of the DIDS effect, changes.

compared

higher

to 6.5,

the extract have

become

Vol. 76, No. 2, 1977

The modulation narrow

pH range

ghosts

and extract)

ion

of anion

around

of the line

broadening

the peak value Cl-

by DIDS.

of the pH peak

reported

for

pH range

studied

representing

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

represents

transport

correspondence

BIOCHEMICAL

Cl-

transport is

probably

non-specific The degree

Cl of line

for

to the

binding

by band

broadening

to be in approximately

binding

site

inhibit

anion

to displace

of line

per band transport Cl-

from

of Cl-

in binding

cannot

of Cl-

on band

both

and that over

of anion membrane

the whole transport, components.

be used to directly

determine

The DIDS-binding

on the other

to band

3 protein(22,24).

by DIDS can be attributed

We suggest

with

by the approximate

broadening

3 and other

to a

to the inhibit-

binding

inhibition

binding.

that

the human red blood sites

supported

line

(seen

be related

a 1 to 1 ratio

broadening

3 monomer.

is

restricted

the peak

might

conclusion

unrelated

hand,

the modulation

that

The remaining

or stoichiometry

Thus

Thus

DIDS-modulation

the affinities is known

7.2.

binding

This

(23).

of

by DIDS is

cell

the is

3, the anion

capacity related transport

to one DIDSof DIDS to to its

capacity

protein.

ACKNOWLEDGEMENTS: The authors would like to thank Mr. Thomas Lew for carrying out the NMR Measurements, Mr. A. Gaarn for his technical help in preparing the samples, and Dr. P. Rnauf for valuable discussions. This study was supported by the Medical Research Council (Canada) by Grant # MT4665. REFERENCES: Cabantchik, Z.I., and Rothstein, A. (1972) J. Membrane Biol. 10, 311-330. Cabantchik, Z.I., and Rothstein, A. (1974) J. Membrane Biol. 15, 207-226. Passow, H., Fasold, H., Zaki, L., Schuhmann, B., and Lepke, S. (1974175) in Biomembranes, Structure and Function (Gardos, G., and Szasz, I. eds.) p. 197-214 (Proceed. 9th FEBS meeting, Budapest). 4. Rothstein, A., Cabantchik, Z.I., Balshin, M., and Juliano, R. (1975) Biochem. Biophys. Res. Commun. 64, 144-150. 5. Wolosin, J.M., Ginsburg, H., and Cabantchik, Z.I. (1977) J. Biol. Chem. (in press). 6. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617. 7. Passow, H. (1969) in Progress in Biophysics (Butler, J.A.V., and Noble,D. eds.) 19, 425-467, Pergamon, New York. 8. Gunn, R.B., Dalmark,M.,Tosteson, D.C., and Wieth, J.O. (1973) J. Gen. Physiol. 61, 185-206. M. (1976) J. Gen. Physiol. 67, 223-234. 9. Dalmark, 10. Stengle, T.R., and Baldeschwieler, J.D. (1966) Proc. Nat. Acad. Sci. USA, 55, 1020-1025. 11. Norne, J.E., Hsalmarsson, S.G., Lindman, B., and Zeppezauer, M. (1975) Biochemistry, 14, 3401-3408. 12. Norne, J.E., Lilja, H., Lindman, B., Einarsson, R., and Zeppezauer, M. (1975) Eur. J. Biochem. 59, 563-473. 1. 2. 3.

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Vol. 76, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

13. Yu, J., Fischman, A., and Steck, T.L. (1973) J. Supramol. Struct. 1, 233-247. 14. Dodge, J.T., Mitchell, C., Hanahan, D.S. (1963) Arch. Biochem. Biophys. 110, 119-130. 15. Shami, Y., Ship, S., and Rothstein, A. (1977) Anal. Biochem. (in press). 16. Fukuda, M., and Osawa, T. (1973) J. Biol. Chem. 248, 5100-5105. 17. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 18. Aminoff, D. (1961) Biochem. J. 81, 384-392. 19. Rothstein, A., Cabantchik, Z.I. and Rnauf, P.A. (1976) Fed. Proc. 35, 3-10. 20. Clarke, S. (1975) J. Biol. Chem. 250, 5459-5469. 21. Cabantchik, Z.I., and Rothstein, A. (1974) J. Membrane Biol. 15,227-246. 22. Ship, S., Shami, Y., Breuer, W., and Rothstein, A. (1977) J. Membrane Biol. (in press). 23. Brazy, P.C., and Gunn, R.B. (1976) J. Gen. Physiol. 653, 583-599. 24. Lepke, S., Fasold, H., Pring, M., and Passow, H. (1976) J. Membrane Biol. 29, 147-177.

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