Reconstitution of band 3, the erythrocyte anion exchange protein

BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RECONSTITUTION

OF BAND 3,

THE ERYTHROCYTE ANION Alonzo

H. Ross and Harden

Stauffer Laboratory Stanford University, Received

December

EXCHANGE PROTEIN M. McConnell

for Physical Chemistry Stanford, California 94305

7,1976

SUMMARY: Band 3, the erythrocyte membrane protein thought to be responsible for anion transport, was purified to near homogeneity using a Concanavalin A affinity column. Band 3 was then combined with egg lecithin, erythrocyte lipid, cholesterol, and glycophorin, the major erythrocyte sialoglycoprotein, to form vesicles capable of The transport activity was sensitive to rapid sulfate transport. prior treatment of the erythrocytes with pyridoxal phosphate-NaBH4, a potent inhibitor of anion transport in these cells.

Studies

INTRODUCTION: suggested

that

protein,

is

hypothesis a crude cyte for

band

with

3, a 100,000

responsible

for

rich

structure

band

a lectin

affinity

combined

it

vesicles

capable

transport

Abbreviations:

column

with

to treatment

in band

3 have

other

by pyridoxal in

intact

weight anion

see ref.

protein

human

red

sulfate

band blood

4 for

(5,6,7,8). 3 to near cell

transport.

phosphate-NaBH4,

integral

(1,2).

This

a review

with of erythro-

Several

methods

We have

employed

homogeneity,

and

(RBC) components The activity a potent

have

membrane

of transport

nomenclature).

been reported

of transport

transport

reconstitution

3 (3;

to purify

of rapid

molecular

by the

and membrane

isolating

inhibitors

erythrocyte

was strengthened extract

specific

to form is

inhibitor

sensitive of anion

RBC (9).

amount of protein dissolved A-U., absorbance unit, 1 ml buffer which would give an absorbance of 1.0 278 nm; DTAB, dodecyl trimethyl ammonium bromide; RBC, red blood cell.

Copyright 0 1977 by Academic AN rights of reproduction in any

Press, Inc. form reserved.

1318

ISSN

in at

0006-291X

Vol. 74, No. 4, 1977

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS: Egg lecithin was prepared by the method of Singleton et al. (10). RBC lipid was isolated from ghosts by successive extraction with 2:l chloroform methanol, 1:2 chloroform methanol, and 7:l chloroform methanol saturated with 28% ammonium hydroxide. Non-lipid material was removed with the Folch washing procedure (11). Glycophorin prepared by the method of Marchesi and Andrews (12) was the gift of E. J. Luna and J. T. Lewis. Dodecyl trimethyl ammonium bromide (DTAB) was synthesized according to Hong and Hubbell (13). Phosphate was assayed by the method of Ames (14), cholesterol by the method of Zlatkis and Zak (15), and protein by the Lowry method (16) (adding 0.1 ml 4% SDS to each tube). Bound pyridoxamine phosphate was assayed by its fluorescence (9). a-Methylmannoside from Pfanstiehl Laboratories was used because of its low absorbance at 278 nm. Sodium dodecyl sulfate polyacrylamide electrophoresis gels were run and stained according to Steck and Yu (17). Erythrocyte ghosts were prepared from outdated RBCs (type 0' from the Stanford Blood Bank) (18). Prior to lysis some of the cells were treated with pyridoxal phosphate and NaBH4 using 20 mM pyridoxal phosphate and 100 mM NaBH4 (9). Polyacrylamide gel electrophoresis was used to check for proteolysis by the NaBH4. Following lysis, the ghosts were washed twice with 3 vols of 15 mM histidine pH 6.6 and then lyophilized. The freeze-dried ghosts were resuspended at lo-15 mg/ml in 100 mM DTAB, 1 mM dithiothreitol, 15 mM histidine pH 6.6, and vigorously stirred for l-l.5 hrs. Insoluble material was removed by centrifugation at 67,500 xg for 30 min. The white pellet was discarded, the yellow supernatant was mixed with a concentrated divalent cation solution. The resulting mixture contained 1 mM CaC12, 1 mM MgC12, 1 mM MnC12. Band 3 was purified by affinity chromatography using a Pharmacia Concanavalin A Sepharose 4B column similar to that used by Findlay (8). Approximately 2 ml of column material was equilibrated with 100 mM DTAB, 1 mM CaC12, 1 mM MgC12, 1 mM MnC12, 15 mM histidine pH 6.6 in a 5 ml pipette. The extract from 20-40 mg ghosts was added to the column followed by a 25 ml rinse with the same buffer. The protein was We collected eluted with 100 mM a-methylmannoside in the same buffer. 2.7 ml column fractions and measured protein content by absorbance at 278 nm. The column was shielded from light during separation of pyridoxal phosphate-treated band 3. Band 3 was reconstituted by a method closely analogous to that of Hong and Hubbell (13) for rhodopsin, and Grant and McConnell (19) for glycophorin. Egg lecithin and RBC lipid at a molar ratio 86:14 were dried from ethanol with a stream of Ar and then placed under vacuum The lipid and lyophilized glycophorin were disfor several hours. Cholesterol dispersed in 100 mM DTAB and solved in column buffer. eluted band 3 were added to give a final composition of 0.79 mM egg 0.13 mM RBC lipid, 0.45 mM cholesterol, 71 pg/ml glycophorin, lecithin, The molar ratios for band 3, glycoand 0.005 A.U. band 3 (Q4 Vg)/ml. phorin, and total lipid were 1:27:29,000. The solution was incubated dialysed against on ice under Ar for 4-5 hrs, and then exhaustively 10 mM sodium phosphate, 2 mM NaCl, 2 mM Na2S04, 1 mM EDTA, 0.1 mM To avoid lipid oxidation, the dialysis buffer dithiothreitol pH 6.6. During dialysis, solutions was continuously aerated with nitrogen. containing pyridoxamine phosphate-band 3 were shielded from light. Lipid protein aggregates were eliminated by 2 or 3 10 min centrifuThe vesicles were harvested from gations (1325 xg) following dialysis. the low speed centrifugation supernatant by centrifugation at 60,000 xg for 45 min. were suspended To study uptake of radioactive compounds, vesicles

1319

BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FRACTION

Figure

1.

NUMBER

Elution profile of band 3 from Concanavalin A affinity The three arrows indicate the addition of the column. solubilized ghosts, the rinse buffer, and the cr-methylmannoside solution.

in dialysis buffer (~2 mM phospholipid) with the radioactive compound, at 23 "C, and at suitable intervals 10 ~1 aliquots were diluted with 5 ml 14.4 mM sodium phosphate, 2 mM NaCl pH 6.6, passed over a 0.22 or 0.45 u Millipore filter, and then washed on the filter with an additional 5 ml solution. The filters were dried and then counted in 10 ml Aquasol (New England Nuclear). Filters were washed with H20 and soaked in the wash buffer prior to use. Incorporation of the protein into the vesicles was checked on a 6 cm 5-15% linear sucrose gradient. The samples were layered on the top of the gradient and centrifuged at 297,000 xg for 7-12 hrs. The protein and lipid content of fractions collected from the bottom of the tube were assayed using the Lowry Assay and 14C-phospholipid. RESULTS: affinity

chromatography

contaminating protein

retained (Fig.

protein has been

by the 1).

This

assigned

figure

gel (Fig.

to the

3 assuming

band

by lectin

3 with

very

solubilized

was eluted

of the

detergent 88,000

little

ghost with

ct-methyl-

by dialysis

daltons

as the

represents

an upper

limit

to the

since

3 itself

might

be phosphor-

band

electrophoresis 2).

band

of total

A column

removal

phospholipids

>90% pure

fraction

band

followed

homogeneous

Concanavalin

per

Polyacrylamide is

nearly

Following

weight.

of contaminating

of RBC ghosts

The small

2-7 phosphates

molecular

ylated.

gave

lipid.

mannoside we found

DTAB solubilization

showed that

The minor 3 dimer

1320

(7).

high

molecular

The identity

the

number

eluted

weight

band

of the

con-

Vol. 74, No. 4, 1977

Figure

2.

taminants

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Gel electrophoresis of column eluate. The eluate was dialysed against distilled water and then 0.1% SDS prior to electrophoresis in order to eliminate the DTAB. The gel shown in the upper tracing was stained with Coomassie Blue and the lower gel with periodic acid-Schiff reagent. The right edge of the trace represents the leading edge Roughly of the tracking dye, and the bar is 0.1 A.U. 20 pg protein was applied to each gel.

varied

with

the preparation.

Glycophorin

and band 4.2 were

by dialysis

of

the most common. Lipid

protein

vesicles

During

the dialysis

peared

and had to be eliminated

vesicles

in the

large

were formed

supernatant

aggregates

of

lipid

the DTAB.

and protein

by low speed centrifugation.

contained

1321

16-36% of

the

total

also

apThe

phospholipid

BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

20. zN iI,. !--&Oa-

<00 tL+ Oo FRACTION 8

4

Figure

3.

Sucrose gradient Percent of total Assay and percent

12

16

20

NUMBER

profiles of reconstituted vesicles. b the Lowry protein was measured of total lipid with 1 i C-phospholipid.

30 TIME

Figure

4.

cholesterol

protein

into

content

tein

Lipid

were nearly

vesicles all

could

but

in

little

be loaded

with

all

formed the

tried

with

mole

was verified

Nearly

We originally alone,

35

40

45

N)

was 23-28

the vesicles

ultracentrifugation. 3).

(MI

Uptake of SO4' (01, sucrose (A.), and Na+ (0) by the reconstituted vesicles. Each point for SO4' uptake represents an average of two runs.

and the

(Fig.

0

top

by sucrose

of in

the the

S04=,

protein same

density banded

manner

but

of

gradient with

the

lacking

lipid pro-

fraction.

to reconstitute success.

The incorporation

%.

band

Even though

egg lecithin

1322

band

3 with

egg lecithin

egg lecithin 3 vesicles

vesicles gave

low pro-

BIOCHEMICAL

Vol. 74, No. 4, 1977

tected

volumes

method or the

(O-O.7

pl/mg

ion exchange

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phospholipid)

with

column method of Michaelson

The problem

was not

caused by the vesicles

since

filters

retained

0.22~

cholesterol,

phospholipid.

effective

than

shows that data

The halftime

by the

vesicles

observing sulfate

to the very

sucrose

degree.

for

Ghosts 4.4 million

prepared

prepared

doxal intact

volume

the

These

was

retained

of uptake

shows that since

nonspecifically

find

4

vesicles

of radioactivity

the completion

internal

Fig.

or Na+.

glycophorin

it

was

we are

is unlikely

to the membrane

endpoint

4 we calculated

for

Na' due to its

the Na+ uptakes

with

control

using

pyridoxal

using

the

about

per ghost.

phosphates

60% greater

band 3 (average

The purified

per band 3 molecule.

of

than

3 pairs

that

band 3 took for

a band 3 associated

a potent

The transport inhibitor

erythrocytes. 1323

is

vesicles

of reconstitutions). sulfate

homogeneous band 3, glycophorin,

and egg lecithin.

phosphate-NaBH4,

molecules

RBC contained

phosphate-NaBH4-treated

We have observed nearly

phosphate-treated

pyridoxamine

a halftime

DISCUSSION:

cholesterol,

the

was more

we have carried

The experiment

phosphate

1.5-2.5

with with

transport

following

from pyridoxal

pyridoxamine

prepared

up sulfate

by lipid

would bind

In Fig.

to 1.5-

sulfate.

band 3 contained Vesicles

reconstitutions

filter vesicles

components

sucrose

(20).

of RBC lipid,

volumes

three

than

the fraction

We did not

slow uptake.

endpoint

into

and sucrose

same

five

and sulfate.

uptake

with

any one or two components.

that

times

the

the addition

of all

up much faster

found

through

the protected

SO4' uptake

at long

actual

of

of the

for

We also

the same for

that

the addition

S04= was taken

25-30 min.

raised

that

The addition

are representative

out.

We found

and glycophorin

6.0 pl/mg

passinq

filter

and Raftery

80-95% of the radioactivity

14C phospholipid.

containing

the Millipore

RBC lipid,

sensitive

of anion

anion

transport

to pyriin

Vol. 74, No. 4, 1977

At present vesicles the

it

with

for

sulfate

transport

transport data

the

per

molecule 0.27

are

sulfate

for

sulfates/set/band

volume

specific

interaction

may stabilize specific

through

membrane

interaction

complex --et al.

suggested (23)

activity

strengthens

in

found in the

and provides

(21) (2)

of

solution.

phos-

have used

we

Ho

to make a rough Using

0.07

of

Since

RBC per ml packed a flux

by the

study

reported.

our vesicles.

lOlo

of uptake

No complete

similar

their

com-

values

RBC and lo6

band

phosphates/set/band

Our experimental

the

other

RBC components

a nonspecific

with

the

quite

rate

3

3

result

is

3 molecule.

know whether

protected

RBC.

transport

flux

a 2 mM phosphate

the

has been

on phosphate

RBC, we calculate

We do not

tein

in an intact

Vmax and Km and assuming

molecules

port

to compare

of

and Guidotti's with

difficult

activity

and sulfate

parison

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

saturation

phate

is

BIOCHEMICAL

band

3.

that

and reduce

da Silva

a whole

a good

the

protein

of band system

(band

was required

for

the

or through negative

charges A

3) (glycophorin) (22).

Also,

to reconstitute extract.

Gasko trans-

Our study

3 as an anion

studying

a

permeability.

and Nicolson

RBC ghost

identification

large

nonspecific

with

RBC lipid

effect

Glycophorin's

is consistent by Pinto

physical

increase

transport

interactions

pro-

between

RBC components. ACKNOWLEDGEMENTS: We are grateful to Drs. R. D. Simoni and W. H. Huestis for many helpful discussions and for the use of their equipment. Alonzo Ross is the recipient of a National Science Foundation Graduate Fellowship. This research has been supported by the National Science Foundation Grant nos. BMS 75-02381 and BMS 75-02381 AOl. It has benefited from facilities made available to Stanford University by the Advanced Research Projects Agency through the Center for Materials Research. REFERENCES 1.

Cabantchik, 2. I. PPS- 207 and 227.

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Ho,

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M. K. and Guidotti,

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A.

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Biol.

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250,

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Rothstein, A., Cabantchik, 2. I., Balshin, M., (1975) Biochem. Biophys. Res. Comm. 64, 144.

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Steck, T. L., Weinstein, D. F. H. (1970) Science

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Passow, H.

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Gasko, 0. D., Knowles, A. F., Shertzer, H. G., and Racker, E. (1976) Anal. Biochem. 72, 57.

V. T. and Andrews,

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