Erythrocyte membrane proteins and adenosine triphosphatase activity

BIOCHEMICAL

Vol. 55, No. 3,1973

AND BlOF’HYSlCAl

RESEARCH COMMUNICATIONS

ERYTHROCYTEMEMBRANEPROTEINSANDADENOSINE TRIPHOSPHATASEACTIVITY Yohtalou

Tashima

Department of Biochemistry, Saitama Medical College Moroyama, Iruma-gun, Saitama, 350-04, Japan Received

October

9,1973

Erythrocytemembranewas treatedfromoutside with crude mold prot ease, and the membrane proteins were analyzed on acrylamide-gelelectrophoresis inthepresence of sodiumdodecyl sulfate. Band-ID protein, in the region of an intermediate of Na-K-ATPase, was digested almost completely, although approximately 80% of the ATPase activity was retained by the treated membranes. It is concludedthatband-ID protein is not the Na-K-ATPase intermediate, although they co-electrophorese. SUMMARY

A number of

INTRODUCTION

investigators’-’

of acrylamide-gelelectrophoresis membranes to develop the

lipid

layer

membrane, band-III to the inner of band-III This

protein During

models

protein5*‘,

the course EC 3.6.1.3),

labeled

[y-“PJATP,

radioactive band-ID

the

from the outer

was suggested

Na-K-ATPase

The molecular

of ATPhydrolysisby apeptide

Na-K-ATPase(ATP

component

in the presence

of the

*y6.

of Na+ and Mg++ 7.

is detectedonthe

digestion

band-III

of the

region

erythrocyte

is attacked protein

used is:

Copyright 0 1973 by Academic Press, Inc. AI1 rights of reproduchbn in any form reserved.

is

one of

the

The of the

membrane With milder

selectively”,

SDS, sodium dodecyl 630

phos-

enzyme can be

and

components

molecule.

*The abbreviation

weight

to 105,000

of the ATPase and loss of structureg.

that

of the

membrane surface

to be 89,000

digestionconditions,band-ID protein it

in

polypeptide3.

Trypsin

causes inactivation

proteins

One ofthemajorproteins

of the ce113p<

phosphorylatedpeptide protein’.

results

solubilizederythrocyte

arrangementofthe

has been reported

phohydrolase with

for

extends

membrane surface

is a single

of

inSDS*

of the membrane.

protein

have used the

sulfate.

of

Vol. 55, No. 3,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

;0, ‘f LOH 36,000 0’ i x 5 GluOH zz 53,ooo

‘-

-L

Origin El

A

Electrophorogram of erythrocyte membrane proteins, from Fig. 1 erythrocytes treated(A) and not treated(B) with"lytic enzyme-3". Membrane sampleswereprepared and electrophoresed in 0.1% SDS on 7.5 % acrylamide gel as described in MATERIALS AND METHODS. The gel was loaded with about 1OOug of membrane protein. Gel were scanned at 570 nm after staining. Arrows show the mobility of proteinswithknownmolecularweight; cytochrome c(Cyt.C),glutamate dehydrogenase(GluDH), lactate dehydrogenase(LDH) andphosphorylasea(Phos-a). In an attempt

to

membrane,

proteaseswere

possible

to digest

Na-K-ATPase MATERIALS

separate

the

three

plasma times

intact

the

erythrocyte

membrane.

It

significant

was

loss

activity. Crude

The crude

enzyme

and acetone

and buffy with

enzyme

containing

proteases

was purchased

MEMBRANE PREPARATION. the

from

band-IUproteinwithout

B-1,3-glucanase,"lyticenzyme-3':

fractionation

enzymes

appliedtothe

AND METHODS

(Tokyo).

the

coat

&155M

was obtained

fromKyowaHakkoCo. by ammonium

fractionation

from

Human blood

(50ml)was

were sodium

removed. phosphate

631

and

sulfate

mold. centrifuged,

The cells buffer,

and

were washed

pH7.4,

and

of

BIOCHEMICAL

Vol. 55, No. 3, 1973

0 “Lyilc

AND BIOPHYSICAL

20

40

enzyme-3”

fig/ml

60

RESEARCH COMMUNICATIONS

80

preincubatlon

mliture

Fig. 2 Effects of hydrolysis of membrane preparation on ATPase activity. Erythrocyte membrane suspension(3.2mgprotein/ml) was mixed with 60ulof "lyticenzyme-3"(0-.8Ong),and incubated for 20 minutes at 37', followed by centrifugation at 156,SOOxg for 30 minutes. The precipitate was suspended in 1 ml of 1 mM EDTA,pH 7.0. 0.1 ml of each was used for the ATPase assay. The assay method is described in the legend of Table 1.

finally

suspended

cells at

in

40mlof

the

(10ml)weretreatedwiththe"lytic 37'.

with

the

The treated phosphate

were performed isolated

protein

membranes

per

cells

were

buffer.

of

The washed

with

3 h

and washed once

and membrane

twice

mg) for

preparation

co-workers".

The

O.SmM EDTA-Tris,

solutiontogivel-2

pH 7.5,

mgofmembrane

ml. OF MEMBRANE.

an equal

of 2% SDS solution

volume

and incubated

in boiling

SDS ACRYLAMIDE-GEL

blue

The membrane

water

suspension

containing 2 minutes.

ELECTROPHORESIS.

The

Calibration

curves

weightswithdisulfidebonds was used

for

the

staining

632

was added

0.4% mercaptoethanol,

for

employed.

known molecular

brilliant

off

Dodge and his

EDTA-buffer

SOLLJBILIZATION

0sborn"was

centrifuged

Hemolysis

were washed

in the

buffer. enzyme-3"(1

by the method

and suspended

of

phosphate

method

of Weber

were obtainedwith reduced. of protein.

and

proteins Coomassie

BIOCHEMICAL

Vol. 55, No. 3,1973

Table

1.

Effects

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Hydrolysis

Pretreated

of

Intact

Cells

on ATPases.

Activity Na-K-ATPase Mg-ATPase

with

(pmoles

Pi/mg/h)

None “lytic

enzyme-3”

0.34 0.31

0.85 0.82

None “lytic

enzyme-3”

0.46 0.32

0.73 0.88

None pronase

0.28 0.30

0.27 0.30

None trypsin

0.34 0.06

0.25 0.14

ATPase activity was assayed in a final volume of 1 ml, containing 14OmM NaCl, 14mM KCl, 5mM MgClz, O.SmM EDTA, 3mMATP, and SOmMTris buffer, pH 7.5, in the presence or the absence of 0.2mM ouabain Incubation was for 1 hr at 37“. Mg-ATPase: the activitymeasured with ouabain, Na-K-ATPase: activity withoutouabainminus activity with ouabain.

INORGANIC deproteinization assay

step,

medium

of Fiske

after

the

(Xlml

for

presence

of

of

followedby

determination

large

eliminate

was added

analysis

No turbiditywas

the

to

5% SDS solution

incubation,

and SubbaRow13.

was successful in

In order

PHOSPHATE DETERMINATION.

inorganic

amountsofprotein

to

each

by the method

observed. of

the

This

method

phosphate

even

(Tashima,

manuscript

in

preparation). PROTEIN

DETERMINATION.

was used. Pi per

The method

The specific

mg protein

per

activity

of Lowry of ATPase

and his

co-workers14

was defined

as umoles

hour.

RESULTS AND DESCUSSION

After

treatment

of intact

erythrocytemembrane

fractionwas

by SDS-acrylamide control

sample

viously1,2,5. the

treated

cells

gel gave

the

prepared,

approximately and -IV

pattern

disappeared most

633

the

and analyzed

As shown in Fig. same

Band-awasthe

"lyticenzyme-3",

solubilized,

electrophoresis.

Band-ID samples.

with

1, the

as reported almost

prominentofthe

completely

prein

new bands.

BIOCHEMICAL

Vol. 55, No. 3,1973

Its

molecular

-V were not

weight

was approximately

changed

remarkably.

distinct

bands in the

that

the

proteins

with

molecular After

region

weights

as shown inTable on K+ was not

The pretreated

than

and -V.

were

digested

1.

with

averaged

remarkably

It

no

appeared

to peptides

the “lytic

enzyme-3”,the

80% of the

The p-nitrophenylphosphatase

changed

and

band-V.

of erythrocytes activity

-II

sample yielded

bands-II

and -IV

smaller

Na-K-ATPase

Bands-I,

43,000.

between

on bands-ID

treatment

remaining

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

control

values,

activity

by the pretreatment

depending (Data

are not

shown). The average Cl8units

specific

activity

of Na-K-ATPase

per mg membrane protein.

Band- III protein

or 30% 5 of the whole membrane proteins specific

activity

stained

of the ATPase as counts

2le.

ATPase,

specific

If

all

of band-III

digestion

inthecharacteristics didnotchange

of band-III If

the effects

solution

and did

to be unrelated

the Na-K-ATPases

III protein quantity

would

would would

alter

it

constitute

is l6

of Na-K-

enzyme-3”

or of ouabain

stabilityofthe

ATPase

NaI. Thus, themajor

component

to the Na-K-ATPase. kidney

activity,

can be estimated

be responsible

protein

The “lytic

the

fromerythrocyte, specific

The

cause some change

K’concentration

(pH 8.5) or in 1.6M

appears

SOO’5T18),

not

on the gel.

were a component

protein

of Na+and

assumed to have similar (above

protein

this

15% 2

activityofkidney’5,

of the ATPase activity.

on the ATPase activity, in alkaline

of

represents

per mg band-III

less than 5.6, WhichislowerthanNa-K-ATPase or brain”

was from Cl3 to

for

and brain

as counts

that

less

permgprotein

than

l.1 %of the band-

the ATPase activity.

a negligible

band on the

are

This

SDS-acrylamide

gel electrophorogram. In

contrast

to

membrane preparationswas

the

intact foundto

cells,

digestion

cause a rapid

634

loss

of

erythrocyte

of Na-K-ATPase

BIOCHEMICAL

Vol. 55, No. 3,1973

activity, gel

as shown in Fig.

AND BIOPHYSICAL

2, and a loss

was observed of pronase

-IV

under were

inactivated

bands

similar

on the

ACKNOWLEDGMENTS

invaluable

faces

the

1).

The effects

of the"lyticenzyme-3".

strongly.

might

toproteases

(See Table

In

all

The resistance

proteases protein

conditions

to those

were attacked.

K-ATPase

her

surfaceoferythrocytes

identical

Na-K-ATPase

nonspecific

for

distinct

electrophorograms. The specificityoftheouter

and

of

RESEARCH COMMUNICATIONS

suggest outside

cases,

of the that

a very

of the

cell.

The auther

is grateful

technical

assistance.

to Miss

Trypsin mainly

ATPase small

Nobuko

bands-III activity area

to of Na-

Yoshimura

REFERENCES

Trayer H.R.,Nozaki Y.,Reynolds J.A.,Tanford C., J.Biol.Chem., 246, 4485 (1971) Bretscher M.S., J. Mol. Biol., 58, 775 (1971) : Bretscher M.S., J. Mol. Biol., 59, 351 (1971) D.M. ,Glossmann H., J. Biol. Chem., 246, 6335 (1971) 4 Neville 5 Wallach D.F.H., Biochim. Biophys. Acta, 265, 61 (1972) 6 Steck T.L.,Fairbanks G.,Wallach D.F.H., Biochem., 10, 2617 (1971) 7 Bader H.,Post R.L.,Bond G.H., Biochim. Biophys. Acta, 150, 41 (1968) G., Proc. Natl. Acad. Sci. USA, 69,1216 (1972) 8 Avruch J.,Fairbanks G.E., Proc. Natl. Acad. Sci. USA, 58, 991 (1967) 9 MarchesiV.T.,Palade 10 Avouch J.,Price H.D.,Martin D.B.,CarterJ.R., Biochim. Biophys. Acta, 1

291,

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