Peptide heterogeneity in the band 3 protein of rabbit erythrocyte plasma membranes

Vol.73,No.3,

BIOCHEMICAL

1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PEPTIDE HETEROGENEITY IN THE BAND 3 PROTEIN OF RABBIT ERYTHROCYTE PLASMA MEMBRANES E. R. Vimr

and J.

R. Carter,

Jr.,

M.D.*

Department of Medicine Case Western Reserve University Cleveland, Ohio 44106

Received

October

5.1976

SUMMARY: We have examined the band 3 protein(s) of rabbit erythrocyte membranes by a combination of differential extraction and surface labeling methods. Only one major peptide was labeled when intact red cells were exposed to 1251- and lactoperoxidase; this coincided with band 3. When intact cells were exposed to galactose oxidase followed by C3H]borohydride, numerous surface glycoproteins were labeled, one of which clearly coincided with band 3. Differential extraction with lithium diiodosalic late revealed one major band 3 glycoprotein which contained both the 1251- and !I H surface labels and three peptides which were unlabeled; these three peptides are apparently not exposed at the cell surface. The protein

composition

polyacrylamide

gel

examined,

50-60

with

electrophoresis,

and band 3 (1, 2). Schiff

staining

cell

membranes,

this

technique.

galactosyl

of mature

per

cent

is

However,

residues

with

gels,

we have Gahmberg

(3)

t3Hlborohydride

and galactose

with

acid

staining.

tance

and because

erythrocyte

interest it

membrane

with

this

Grant

*To Whom Reprint AM 18389.

Copyrighr All rights

glycopeptide

has focused

is a glycosylated (4,

5).

Several

(6-8).

There

Requests

Should

0 1976 by Academic Presr, Inc. of reproductim in any form reserved.

with

membranes

(2);

specific

which

has been disagreement

Be Addressed.

779

rabbit

oxidase,

that

than

red

glycoprotein labeling

spans

functions

acid-

specific

on band 3 due to its peptide

so far molecules

by periodic

show no major

by

species

two "spectrin"

as assessed

has shown,

in human erythrocyte

Considerable

mammalian

of the

been working,

are present Schiff

in all

as demonstrated

is much more variable

glycoproteins periodic

membranes,

composed

composition,

of polyacrylamide which

cell

similar

by weight

Glycoprotein

with

red

by of

many more

can be observed

quantitative the

full

imporwidth

of the

have now been associated as to whether

Supported

in part

this

by NIH

Vol.73,No.3,

1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

band represents

diffuse

or several

co-migrating

band 3 in rabbit surface-exposed molecular

a single

(10).

species

erythrocyte peptide

weight

peptide

which

species We report

with

variable

glycosylation

here experiments

membranes is composed of at least

and three

nonglycosylated

are apparently

peptides

that

one glycosylated,

of closely

not exposed at the cell

Materials

indicating

(9)

similar

surface.

& Methods

Materials: Galactose

Lithium diiodosalicylate was obtained from Eastman Kodak Co. oxidase and lactoperoxidase were from Sigma Chemical co. Carrier-free 1251- (11-17mCi/pg) and sodium [3H]borohydride (lOCi/mol) were from Amersham/ Searle. All other reagents were reagent grade.

Preparation and Labeling of Red Cells: Rabbit blood was collected from an ear vein into a heparinized beaker. Red cells were washed three times in NKM-PO"1 buffer. For galactose labeling, washed packed cells were diluted in 5 volum& of NKM-PO buffer and treated essentially as described by Gahmberg (3). To 0.5ml of ccl 4 suspension at 37" was added 12.75 units of galactose oxidase; the cells were incubated for 1 hour with intermittent gentle shaking, then washed twice with NKM-PO4 buffer and resuspended in 0.5ml of the same buffer at 37". Reduction of galactose residues was performed for 30 minutes with the addition of 1mCi of [3H]borohydride. The reaction was stopped by the addition of 10 volumes of ice cold NKM-PO4 buffer and the cells washed three times. Only terminal galactose incubated without galactose residues are labeled with this method, Controls oxidase incorporated only l-2% as much 3H as cells exposed to the enzyme.

Intact erythrocytes suspended surface with 1251- in the presence by Morrison (11).

in NKM-PO buffer were labeled of peroxi d ase and H202 exactly

on the external as described

red cell Preparation and Gel Analysis of Membranes: After labeling intact cells, rglost" membranes were prepared as described by Cabantchik and Rothstein (8). Membrane proteins were fractionated in sodium dodecyl sulfate and dithiothreitol on 5.6% polyacrylamide gels as described by Fairbanks et al (l), stained with Coomassie Blue, and scanned at 580 nm with an Isco model 659 gel scanner. The gels were then sliced at 1 or 2 mm intervals in a Mickle Gel-Slicer. 1251 in each slice was determined directly in a well-type gamma counter. To determine 3H incorporation, slices were dissolved in H202 and radioactivity determined in a toluene-based fluor in a liquid scintillation counter. Results When rabbit borohydride

erythrocyte

and analyzed

membrane glycoproteins by polyacrylamide

peaks were observed;

the majority

and did not coincide

with

1) 3.75fl

& Discussion

specific

gel (Fig.

were labeled

with

tritiated

1)) a number of radioactive

of these were at a molecular Coomassie Blue-stained

weight

peptides.

-z 90,000 The single

The abbreviations used are: NKM-PO4 buffer: 115mM NaCl, 3.75mM KCl, MgC12, 3OmMphosphate buffer pH 7.5; LIS, lithium diiodosalicylate.

780

BIOCHEMICAL

Vol. 73, No. 3,1976

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

1.

2 0 012345678 Migration

(cm1

Fig. 1 Peptide and [3H]galactose profiles of rabbit erythrocyte membrane proteins labeled with sodium [3H]borohydride after oxidation with galactose oxidase. Protein band numbering is according to Koch et al (12). Solid line, profile of Coomassie Blue stained peptides (A5BO); Dottamne, tritium /l mm gel slice.

Table

I.

Extraction

of rabbit

erythrocyte

LIS Concentration (mM)

membranes

with

% Protein Extracted

lithium

diiodosali~ylate.~

% K3HlGalactose Extracted

0 10 30

12 8 2 65 67

1:: 200

1; 43:

aExtractions were performed as described by Furthmayer et al (13). Each extraction with the indicated concentration of LIS was FrKrmed using lmg of membrane protein containing 3.7~106 tritium cpm.

radioactive to band

peak migrating 3 (Fig.

When labeled the amount (Table

I).

not observed

weight

of 100,000 clearly

corresponded

1). membranes

of protein

were

extracted

In contrast, until

at a molecular

extracted

with

increased

a significant

1OOmMLIS was reached

increasing

steadily increase (Table

781

with in

I).

concentrations each

t3H]galactose

increment

of LIS, in LIS

extracted

was

Vol.73,No.3,

1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 2000 t) D 1000

;

0

ii CD -7

L

0

I

,

,

1

2345678

.

I

Migration

(cm)

Fig. 2 Identification of erythrocyte membrane proteins extracted by LIS and their extent of glycosylation. From the experiment described in Table I, equal volumes (50~~1) of control, 10, 50 and 1OOmM LIS extracts were fractionated, stained and scanned on polyacrylamide gels as described in Methods (solid line). In addition, the radioactive profiles of the 1OOmM and 5OmM extracts were determined by counting 2 mm gel slices (broken line). A, 1OOmM LIS extract; B, 50mM; C, 1OmM; D, OmM.

Polyacrylamide difference stained

in the with

region

gel

(Fig.

Coomassie 2B),

of the

band 3 region Blue,

whereas

diffuse

band observed

and the

presence

trailing

analysis

(Fig.

showed

the

membranes

of L3H]galactose

Externally presence

of

coinciding oriented lactoperoxidase

with

small

1OOmM extract

in intact

(Fig.

membrane

and H202;

a striking

this

782

1).

in the

in the band 3

gels

(Fig.

50mM extract

sliced

with 2A); (Fig.

125I

broad,

were

peak coinciding

with

and

the typical

When these

can be labeled technique

separated peaks

1OOmM LIS extract

peaks

proteins

discrete

2A) showed

a large

in the

three

but

(Fig.

determined,

the

revealed

The 50mM LIS extract,

2). three

edge of band 3 was observed

none was seen

LIS extracts

the virtually

28).

in the

has been used to show that

BIOCHEMICAL

Vol.73,No.3,1976

AND BIOPHYSICAL

1 5

10

20

30

Fraction

RESEARCH COMMUNICATIONS

40

No.

Fig. 3 Polyacrylamide gel fractionation of 1251-labeled membrane proteins. A, 50mM LIS extract; B, 1OOmM LIS extract; C, unextracted membranes. The horizontal bars indicate the region of each gel containing stained protein of band 3. Approximately 25ug of protein was fractionated on each gel.

band 3 is

in

technique

with

to examine

fact

glycophorin

is

the

possibility

membranes

also

technique,

PAS-stainable

containing

the

band in rabbit

three

no radioactivity. peak of

by this

discrete On the other

1251-labeled

peaks hand, peptide

is

in

within the

the

783

contrast

membranes

that

with

band 3 region

the

intact

showed (Fig.

this LIS

3). only

one where

the absence

The 50mM LIS extract

in

with

to human membranes,

lOOn#l LIS extract

observed

We combined

3C) indicated

and coincides

membranes.

(5).

in band 3 (Fig.

(Fig,

this

cell

erythrocyte

of heterogeneity

labeled

labeled

of the

of rabbit

band 3, was labeled;

a major

the sharp

surface

extraction

of intact

peptide,

on the outer

differential

further

Electrophoresis major

exposed

(Fig.

of 3A)

essentially 38)

contained

membranes.

Vol.73,No.3,1976

It

is

thus

of at least brane,

is

BIOCHEMICAL

apparent

4 peptide

not

from

peaks

be exposed intact

with

the

which

present

the present

time,

co-migrate

with

transport,

the

port

the

would

be associated

the

the

'Fairbanks,

G.,

Steck,

2Hamaguchi,

H.,

and Cleve,

3Gahmberg,

C.G.,

J. Biol.

T.J.,

SMueller, 6Brown,

P.A.,

Feinstein,

acabantchik,

Z.I.,

IlMorrison,

Meth.

lgcabantchik,

J.

H.,

Z.I.,

Kahane,

I.,

these

appear

smaller

to when At

peptides

the membrane

(14)

water

or simply

In the case of anion would

suggest

that

trans-

functions

and glucose

assigned

transport

Biochemistry

Biophys.

Biol.

Res.

lo,

might

(1971).

2602

459 (1972).

Commun 5,

Chem. -’ 249

and Sha'afi, 1582

A.,

J. Biol.

Enzmol.

dis-

defined

Of the other

R.I.,

32,

Nature

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(1974).

254,

523 (1975).

15,

207 (1974)

9170 (1975).

Chem. 250,

R.,

and Wallach,

D.F.H.,

103 (1974).

Gardner,

F.H.,

and Marchesi,

and Rothstein,

7568

(1976).

H., Bhakdi, S., Schmidt-Ullrich, Biophys. Acta. 330, 356 (1973). M.,

smaller,

510 (1976).

Chem. 251,

12Koch, P.A., Gartrell, J.E., Acta 389, 162 (1975). 'SFurthmayr,

M.,

M.B.,

T.L.,

species is

59-, 351 (1971).

Biol.

and Rothstein,

J., and Steck,

(Q, 75%)

of the glycoprotein.

weights.

D.F.H.,

Biochem.

Chem. 251,

J. Biol.

A.,

laKni.ifermann, Biochim.

H.,

three

mem-

glycoprotein.

and Wallach,

and Morrison,

7Kahlenberg,

gYu,

T.L.,

the cell

and do not

within

itself.

to assume that

are

not clearly

whether

and Rothstein

transmembrane

J. Mol.

M.S.,

are

molecular

glycoprotein

be logical with

peaks

of preponderance

of similar

of Cabantchik

with

there

composed

the majority

residues

to indicate

is

one or more peptide

band 3 glycoprotein

by virtue

is associated

4Bretscher,

no data

with

these

spans

to represent

galactosyl

because

membranes

is glycosylated,

In addition

surface;

is

studies

to band 3, it also

cell

there

it

erythrocyte

represents

studies.

examined

have any association

this

no terminal

to the outer are

rabbit

lOOnit LIS and appears

contain

membranes

3" in

One of these

in band 3; whether

evident

crete

"band

species.

extracted

of the protein

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A.,

and Carter, V.T.,

J. Membrane 784

J.R.,

J. Membrane Biol.

15,

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227 (1974).