Exterior proteins on the human erythrocyte membrane

Vol. 45, No. 4, 1971

BIOCHEMICAL

EXTERIOR

PROTEINS

ON THE

David

13,

ERYTHROCYTE

and Martin

of Biochemistry,

Research

September

HUMAN

R. Phillips

Laboratories

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hospital,

Morrison

St. Jude

Memphis,

MEMBRANE

Children’s

Tennessee

38101

1971

SUMMARY Membranes from pronase treated human erythrocytes were isolated and the membrane proteins se arated by disc gel electrophoresis in sodium dodecyl sulfate. A comparison of pronase treated ccl Ps and untreated cells show that pronase treatment of intact human erythrocytes results in the alteration of three membrane proteins with molecular weights of 90,000, 95,000, 105,000 and reduces their molecular size. All of the proteins with molecular weights of 90,000 and 105,000 were altered indicating that all proteins with these molecular weights must be exposed on the outside of the membrane. Protein fragments formed from proteolytic di stion remain with the membrane, and have an a parent molecular weight of 65,000. The residual ffragments are insensitive to further hydrolysis, an B appear to be portions of the hydrolyzed proteins which is buried within the membrane structure.

INTRODUCTION

Evidence membrane the

from

a number

is asymmetric.

exterior

surface.

neuraminidase

(l),

proteolytic

All

proteins.

From

data Most

sialic

most acid

carbohydrate

(2-4).

membrane,

membrane

investigators

example,

the

while

enzymes

erythrocyte

For

of

have of the

One

exception

is the acetylcholine

appears

reagents

which

react

evidence

that Most

membrane exposed

to be with

on

protein

the

convincing

with

to the

exterior

finding surface

suggesting

treatment

groups,

although

asymmetric

that

the

proteins

cell

not

distribution in

the

removed

in the

of functional

the inside

of the red cell. by

proteolytic

(8,9). have

membrane proteins

Chemical

also

provided

(10,ll). red

cell

class

of 90,000

are

lactoperoxidase

(12).

The

weight by

by

2,3-diphosphoglycerate

definitive,

of

on with

present

membrane

molecular iodinated

be

can be removed

in the erythrocvte

can be catalytically

1103

red

is exposed

activities

(5),

towards

which

of the

erythrocyte

distribution

ATPase

surface

to

can

an asymmetric

activity

human

of erythrocytes

of enzyme

(7) are oriented

assembled

the

determinants

including

regard

and

by

antigenic

esterase

functional

are asymmetrically

recent

removed

activities,

exterior

that

of the membrane

the localization

enzymes

the proteins

is the

of

enzyme

as the glycolytic

and

be

accumulated

(6) as well

enzymes

can

studies

suggested

of the carbohydrate

containing

phosphatase notable

has

in

the

BIOCHEMICAL

Vol. 45, No. 4, 1971

remaining

membrane

iodination

(13).

of the

cell,

proteins,

These

whiie

data

most

however, indicate

can only

that

membrane

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only

proteins

be iodinated

when

a few membrane

are exposed

the

proteins

cell

was

lysed

are exposed

to the hemoglobin

prior

to

to the outside

containing

cell

interior

(13). Pronase amino

acids

is a non-specific (14).

erythrocyte

This

enzyme

membrane

examined

in order

protease has been

surface

(2).

to further

which

will

shown

those

Human cells were

erythrocytes

were

suspended

in 0.106

then

25 pgg/ml pronase periods were

of time, then

(Calbiochem

procedure

was repeated

membranes

were

sodium

a second

isolated

dodecyl

M phosphate

buffer,

time

and

modified

from

the

by pronase

are

of the erythrocyte

0.106

as

at 37OC

described

membrane.

electrophoresed

The

on 5%

&scribed

to a hematocrit constant

at 1,500

Xg

M phosphate

(13).

previously

with

buffer

to free the cells of pronase

as previously

sulfate

proteins

and centrifuged

of the

to their

carbohydrate

proteins

pH 7.4,

and incubated to 4’C,

in 10 volumes

completely

AND METHODS

of serum

cooled

almost

of the

on the outside

free

B grade)

the cells were

suspended

washed

much

membrane

the proteins

MATERIALS

proteins

to remove

In this report,

characterize

hydrolyze

(13).

of 20% containing shaking.

for

After

polyacrylamide

varying

I5 minutes.

and again

proteins gels by

The

centrifuged.

and the hydrolysis

membrane

The

cells This

products.

were

The

solubilized

standard

in

procedures

(13). RESULTS

The

effect

composition

of pronase

is shown

erythrocytes.

The

Identical

protein

60 minutes

Pronase membrane

of

B and

concentrations

is decreased.

Band

molecular

III,

weights

separate

components.

Concomitant

proteins

is an increase

of proteins

pronase

1 represent

alter the

at 37’C

for

the membrane

15 and molecular

molecular

weight

protein

and the

95,000,

decrease

a molecular

1104

weight

respectively, in

the

isolated

published 0.106

from

untreated (13,15).

M phosphate

proteins

from

molecular

buffer

of

between

90,000

removed.

become

identifiable molecular

IV. After

from After

is completely

Band

selected

composition respectively.

weights

for

the membrane.

weights

protein

90,000-105,000

of 65,000,

protein

previously

60 minutes,

with

105,000

with

does

remove

of proteins

the 90,000

with

in the

do not

on the membrane

proteins

to that

cells incubated

however,

C in Fig.

of

is similar

conditions

cells,

25 &g/ml

105,000

with

the

Gels

the

II,

these

with

incubation,

and

that

from

erythrocytes

the membrane

proteins

obtained

indicating

minutes

human

1. Gel A represents

were

treatment

treated

of intact

of separated

patterns

proteins.

erythrocytes

in Fig.

pattern

at 37OC,

treatment

AND DISCUSSION

15 and

Bands

I

as two weight

treatment

of

Vol. 45, No. 4, 1971

BIOCHEMICAL

AND BIOPHYSICAL

---B ?I,a_

RESEARCH COMMUNICATIONS

C

Figure 1 - Acrylamide gels of membrane proteins from normal and pronase treated erythrocytes. Stroma were isolated from untreated erythrocytes, gel A, and from er throcytes treated at a 20% hematocrit with 25 pg/ml pronase at 370C for 15 and 60 minutes, ge r s B and C respectively. The stroma were solubilized in 3% SDS, electrophoresed on 5% polyacrylamide gels containin 0.1% SDS and stained for protein with coomassie brilliant blue. Protein molecular weight bands Habeled correspond to molecular weights of 105,000, 95,000, and 90,000, and 65,000, numbers I, II, III and IV respectively.

the

erythrocytes

for

60

concentration

of

protein

concentration

of

the

minutes,

this

in Band

I, and

other

protein

pattern

is further

a further

molecular

accentuated

increase

weight

in

classes

the are

with

a decrease

intensity not

of

affected

Band by

in IV.

the

the The

pronase

treatment. These

protein

stained

gels shown

weight

regions,>

in protein reduced occur

which

alterations in Fig.

this time

2. No However,

coincides

with

by

I5

an additional

more

alterations

200,000.

in intensity after

are

easily

in protein

in the regions

the increase

minutes

of Bands

treatment,

incubation.

by

the

composition

in the amount

pronase

45 minutes

visualized

Band

densitometer appear

tracings

in the higher

I, II and III there of protein

in Band

however,

no further

I, however,

molecular

is a marked IV. Band changes

is completely

of the

decrease

II, is greatly in this

removed

band during

period. The

present

results

molecular

weights

of

molecular

weights

must

90,000

show and

be on the

that

pronase

105,000. outside

reduces This

the

means

of the membrane.

1105

molecular that

weight

all membrane The

95,000

protein

of all proteins

with

proteins

these

with

molecular

weight

BIOCHEMICAL

Vol. 45, No. 4, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

J L Figure 2 - Densitometer the gels and the numbers

class is only

partially

One

is digested

are

of these identical

different buried

with

proteins:

tracings of the ac lamide gels shown in Fig. 1. The to the protein mo 7 ecular weight classes in Fig. 1.

removed

by pronase

and

one is unaffected

different one

located

in the membrane Band

hydrolytic concentration

with

from

pronase

further

1 and

resulting time

membrane

present

pronase

types

that

these

or alternatively

in the stroma treatment.

treatment by

of two

It is possible

of the membrane

and

correspond

thus

they

of proteins. two

may

digestable

to

proteins

be entirely and the other

to the enzyme.

2 is not

of pronase

hydrolysis

the

on the outside

from

to be composed

by this enzyme.

in

and inaccessible

IV of Figs. product

orientation

and appears

letters

non

1106

untreated

erythrocytes

and is a

Since

this

protein

material

increases

that

this

peptide

fragment

is protected

and

this

it appears

protein

from

membrane

components,

that

lack

in

of

Vol. 45, No. 4, 1971

hydrolysis

is a function The

90,000

new

That

pronase

formed

molecular

this

susceptibility

of the membrane

peptide

to 105,000

lipid.

BIOCHEMICAL

65,000

of

the

fragment

concentration

organization,

in the present weight

peptide

region is not

and not

study

which

to

could

by

further

RESEARCH COMMUNICATIONS

of the protein represent

are buried

covered

fragment

used

AND BIOPHYSICAL

seems

hydrolysis.

intact

erythrocytes

if all the proteins

altered

by pronase

a portion

of proteins

in the membrane

protein

to incubate

structure.

and are covered

evident

Indeed,

due

to the

a twenty-fold

has no effect

in the by

lack

of

increase

on this peptide

of

molecular

weight. It is not

clear

on the membrane

surface.

Lactoperoxidase

labeling

90,000

molecular

of

the

polyacrylamide in this

disc

90,000

susceptible

are indeed

on the

probably protein

and

iodination

Since

this

are in essential

(12).

giving

becomes from

by carbohydrate

class

further

because

surface.

Thus,

lack

results

two

molecular

weight

weight

the

these

of exposed

tyrosines

proteins proteins

surface enzyme

proteins

5%

are

class of proteins

proteolytic

latter

in the by

different

on the membrane

when

components

as estimated

that

this molecular

proteins

solution

of the

proteins

90,000

that

protein

erythrocytes

indicates

These

other

the

the membrane

either

membrane

evidence

to

represent

of intact

evidence

(16).

The

exposed

of

Recent

of the membrane.

carbohydrate

iodination

class are labeled

to pronase

that

lactoperoxidase covered

weight

sruface

protein

weight

gel electrophoresis

molecular

also very

catalyzed

treatment

or more removes

are inaccessible or because

they

to are

or protein.

manuscript

agreement

was completed, with

the data

two

presented

publications

(17,18)

have appeared

whose

results

in this paper.

ACKNOWLEDGEMENTS

The This

work

National

authors was

Institutes

gratefully

supported of Health

acknowledge in part

by

the

grant

capable

number

technical

GM-15913,

assistance DE-02792

of Miss

Becky

Wailer.

and GM-39472

of the

and by ALSAC.

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

E.H. Eylar, M.A. Maloof, O.V. Brody and J.O. Oncley, J. Biol. Chem., G.M.W. Cook and E.H. Eylar, Biochim. Biophys. Acta, 101:57 (1965). SKornfeld and R. Kornfeld, Proc. Nat. Acad. Sci., 63: 1439 (1969). R.J. Winzler, E.D. Harris, D.I. Pekas, C.A. Johnson and P. Weber, (1967). V.T. Marchesi and G.E. Palade, J. Cell Biol., 35:385 (1967). W. Schroter and M. Neuvians, J. Membrane Biol., 2:31 (1970). D.E. Green, G. Murer, H.O. Gultin, S.H. Richardson, B. Salmon, G.P. Arch Biochem. Biophys. 112:635 (1965). B.G. Firkin, R.W. Bed, G. Mitchell, Australian Annals of Med., 12: 26 F.Herz, E. Kaplan and J.H. Stevenson, Nature, 200:901 (1963). H.C. Berg, Biochim. Biophys. Acta, 183:65 (1969).

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

M.B. Bellhorn, 0.0. Blumenfeld and P.M. Gallor, Biochem. Biophys. Res. Commun., (1970). D.R. Phillips and M. Morrison, Biochem. Biophys. Res. Commun., 40:284 (1970). D.R. Phillips and M. Morrison, Biochemistry, 10: 1066 (1971). Nomoto, J,, Biochem. (Japan), 48:598,900 (1960). T. Lenard. Biochemistrv. 9: 1129 (1970). D.R. Philb s and M. Mbrrison, Fed. Prdc., 30, 1065, 1971. W.W. Ben cfer, H. Garan and A.C. Berg, J. Mol. Biol., 58:783 (1971). M.S. Bretscher, Nature, 231:229 (1971).

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