Fatty acid composition of lipids which copurify with band 3

Vol. 159, No. 3, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

1012-1019

Pages

March 31, 1989

FATTY ACID COMPOSITION

OF LIPIDS

WHICH COPURIFY WITH BAND 3 Leonard Department Received

R. Maneri

of Chemistry,

February

Purdue

and Philip

University,

S. Low

West Lafayette,

Indiana

47907

6, 1989

Summarv. In a previous study (L. R. Maneri and P. S. Low (1988) J. Biol. Chem. 263, 16170-16178) we determined that the anion transport protein, band 3, was significantly stabilized by lipids containing saturated and/or long chain fatty acids. To determine whether this thermodynamic preference is reflected in the composition of lipids tightly associating with the anion transporter in vivo, we have analyzed the fatty acid content of phospholipids co-isolating with the purified integral domain of band 3. Our data demonstrate that although stearic acid comprises only 14% of the bulk lipid fatty acids of the red cell membrane, it constitutes -68% of the fatty acids of lipids co-isolating with band 3. Certain other long chain fatty acids were also enriched in the adherent lipids. These results suggest that the fatty acids which most effectively stabilize band 3 also have the highest affinity for the transport protein. 0 1989 Academic Press, Inc. Introduction. into

Spectroscopic

the

effect

behavior the

of

integral

of associated

concept

that

to

may be perturbed

suggest

that

a limited

protein,

One of has

been

to

conditions which

most

per

has

band stability

number

of

(8,9).

desired

nonionic

anionic

1:l

to copurify

with

the

tightly

are

required

for

In a previous

study

of

of

Both

reconstituted band

3 b

oxidase,

the

bound full

thermal

into situ

between

was

Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

lipid

with

the

binding

nondenaturing

analyze

the

glycophorin

high

been

(5)

and

hand,

selectively

affinity

(7),

6 and 10 neutral cardiolipin

lipids

has

phosphatidylserine

stability

approached

1012

data

and

but

phospholipids the

less

avidly

activity.

exogenous

OOD6-291X789 $1.50

of

(5-9).

to

very

to

integral

tightly

on the other

with

phase

has led an

types

under

then

e.g.

stochiometry

very

example,

lipids,

to

preferential

and

For

and

data

Other

manner

insight

dynamics

derived

protein

detergent)

provided

adjacent

establish

integral

phospholipids 3 protein

specific to

Cytochrome

a

of the lipids

may associate

protein.

with

(6). found

methods

the

presence.

lipids

in a highly

the

in

been

monomer

bound

protein's

to copurify

cardiolipin

also

the

in with

phosphatidylinositol binds

by

the

(usually

of

have

on

Analysis layers

direct

purify

copurify

demonstrated

(l-4).

studies

proteins

several

possibly

the

calorimetric

membrane

lipids

one

protein membrane

and

of

lipids, only

the

integral

we demonstrated when

reconstituted

domain that

of the into

Vol. 159, No. 3, 1989

BIOCHEMICAL

zwitterionic

lipids

which

to thicken

served

as increasing

the

also

imparted

head

group

similar

of

which

activity on the

to fatty

chains into

content

stability

that

which

lipids

imparts

Furthermore,

band

3.

the

acyl

While

which

it

copurify

greatest

the anion transporter acid composition of

any

chain

has been with

such

unsaturation, shown

band

thermostability the

factor

3 was reconstituted,

or decreasing

to band the

(10).

that

the

3 (11-13)

is

and

highest

(13, lo), no information lipids which bind tightly

is to

protein. We have

thermodynamic realized

in

nonionic

undertaken

this

preference

for

vivo.

they

band

analysis indeed

study

contain

to

lipids

We demonstrate

detergent,

Compositional that

acyl

cholesterol

greater

to

long

the bilayer

composition

catalytic available this

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3

of the acyl

examine

with here

retains chains

a preponderence

more

long,

that 5

even

after

tightly

bound co-purified

of these of

closely

saturated

the

long

whether acyl

extensive

chain,

the

chains

is

washing

in

phospholipids. lipids reveals saturated

fatty

acids.

Materials and Methods. DEAE silica gel (100 mesh) was purchased from Diagnostic Specialities (Methuchen, N. J., U.S.A.). Ultra pure (gold label) petroleum ether was from Aldrich. The nonionic detergent C1,E, (octaethoxy mono-n-dodecylether) was purchased from Nikko Chemical Co., Japan. All other organic solvents were spectroscopic grade and the chemicals used were reagent grade. Phospholipids were the purest grade available from Calbiochem. Band 3 Durification/delinidation. The band 3 preparation procedure used was the same one published previously for reconstitution experiments (10). Briefly, ghosts were prepared and spectrin depleted to form inside out vesicles, and these vesicles were in turn subjected to limited proteolysis with trypsin to remove the cytoplasmic domain of band 3. The membranes were then dissolved in the nonionic detergent C,,E, (octaethoxy mono-ndodecylether) and the soluble components were applied to an ion exchange column (DEAE Sepharose-6B) where they were washed with -12 column volumes of The fractions the same detergent and eluted with a linear salt gradient. containing band 3 were pooled and subjected to the procedures described below. Lipid extraction. The lipid extraction procedure is a modification of the method of Bligh and Dyer as, suggested by Dr. C. Kent of Purdue (14) In a 30 ml corex centrifuge tube, 2.4 ml of pooled band 3 University. extract in 0.1% C,,E, buffer was mixed with 9 mls of chloroform: methanol During (1:2) to which had been added 50 pg butylated hydroxytoluene. vortexing, 3 mls of chloroform was added and then 3 mls of 0.9% NaCl was introduced to produce two phases. This mixture was vortexed vigorously for 1 minute, placed on ice, then centrifuged 5 minutes at 4,000 rpm in a Sorval The nonpolar phase was carefully removed, HB-4 swinging bucket rotor at 4°C. was aqueous phase and the precipitated protein, which leaving the The aqueous phase was concentrated at the interface between the two phases. extracted one more time by adding 6 mls of methanol and 5.4 mls of 0.9% The 0.01 M HCl and vortexing 1 minute followed by centrifugation. NaCl, nonpolar phases were combined and evaporated under a stream of nitrogen. Phosnholipid purification and transesterification. The detergent was removed This was from the lipid extract by ion exchange chromatography (15). necessary due to the vast difference in the amount of detergent and lipid present since the former often overshadowed the latter in the subsequent The transesterification procedure was taken from (16), and the analyses. 1013

Vol. 159, No. 3, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reaction was allowed to incubate for 24 hours in order to ensure reaction of the longer chain fatty acids. The combined extracts from an original 19.2 mls of purified protein solution were taken up in 2 mls of chloroform and applied to a 2 ml DEAE silica gel column equilibrated in the same solvent. The column was washed with 10 column volumes of chloroform at 0.7 ml/min. and then eluted with chloroform: methanol: 0.8 M sodium acetate (30:60:8). The turbid fractions were pooled and evaporated under a stream of nitrogen, and then were divided and sealed in glass ampules with a total of 5 mls of 5% sulfuric acid a methanol. 2 of each standard mg (dimyristoylphosphat~~ylcholine, dioleoylphosphatidyl choline, and egg yolk phosphatidylcholine) were treated in the same way as the pooled DEAE silica gel eluent. The ampules were then incubated at 70°C for 24 hours and extracted as in (16). Analytical nrocedures. The fatty acid methyl esters were analyzed by gas chromatography-mass spectrometry on a Finnigan 4000 GC/MS with a model 9610 GC and a 3 meter OV-1 packed column at a flow rate of 30 ml/minute. The injection temperature was 25O"C, and the column temperature was programmed to run from 50°C to 300°C. Mass spectra using a source temperature of 250°C and an electron energy of 70 eV were taken of each peak to confirm its identity. The unknown (band 3 lipids) sample was also run on a DB-1 capillary column in order to confirm the identity of an overlapping peak. These confirmatory spectra were run at temperatures from 50°C to 240°C and used an electron impact ionization of 70eV. Peak heights were measured to estimate relative abundances, however, it should be noted that they agreed well with the values obtained using peak areas. Two gas chromatogram/mass spectrograms were taken for the heterogeneous samples (egg lecithin standard and the band 3 associated lipid), one utilizing the chemical ionization (using isobutane at 0.30 torr) and one using electron impact ionization. One spectrum was taken for each homogeneous standard. Confirmation of mass spectra was accomplished by library searches of a computer database and/or direct comparison between the standards and the unknown sample. Lipids were quantitated by the procedure of Rouser (17) and protein concentrations were either derived from the optical absorbance at 280 nm (~-55000 cm-l M-l) or were analysed by the modified Lowry procedure of Peterson (18).

Results Band

3 delinidation.

integral

domain

dodecyl

ether.

(DEAE

Sepharose

lengths

of

removed

The

data

during

3.

resulting band difficult

the

extracted.

preparation band

6B) (at

1 shows

3 in

In this

time

strength. were

Figure of band

the

experiment, column,

was

washed

first

Very

similar

in

a similar

to remove

lipid were

limiting

Unfortunately, the

and

ion

of

which

of

in

extensively column,

of the delipidated polypeptide. For this reason, conducted on a band 3 preparation which was

were

few additonal

extensively

washed

phosphate the

6 tightly

this

at high

lipids

per

detergent bound

Triton

all

ionic easily lipids band mole

3 of

X-100, per

band

3 was

due to aggregation

subsequent

washed

various

phospolipids

delipidated probably

the

exchange

for

eluted

the

lipid-derived

exchange

1014

solution then

of the

obtained

value

of

mono-n-

on an ion

detergent

rates),

of

delipidation

3 was immobilized

content

5 moles

results

from

of

octaoxyethylene

most that of washing, after

15 hrs to be

kinetics detergent

with

flow

demonstrate

found

3 monomer.

band

two different

The residual

the

nonionic

for

studies 16

hrs.

were This

Vol.

159,

No.

3, 1989

BIOCHEMICAL

AND

BIOPHYSKAL

RESEARCH

COMMUNICATIONS

100

75 20 \

18.

16 -. 14 -. 12-.o , o

Lipid/Protein Ratio

8-6 -.

+

0

.

4 -2 -.

01

ot

; IO

O

: 20

: 30

; 40

: SO

Wash

: 60

: 70

: X0

: 90

I 100

0

100 400 500 600 RetentKm Tlnx (FCC,

200

0

2

(hrs)

Time

Fieure 1. Kinetics of delipidization of the integral membrane domain of human erythrocyte band 3 protein during washing with buffer containing 0.1% octaoxyetheylene mono-n-dodecyl ether while immobilized on an anion exchange column. Open symbols were obtained in delipidation experiments at a flow rate of '2 column volumes/hour while solid symbols were obtained at a flow rate of 0.2 column volumes/hour. The vertical lines indicate the standard deviations of the measurements. Where the standard deviation doesn't exceed the size of the symbol, no error bars are shown. Fipure 2. Typical the phospholipids erythrocyte

band

preparation eluted of

from

the

2 shows

isolated noted

on

Although separate

confirm

capillary

characterized

by with

the

retention

domain mass

methyl

esterified

on

easily report in

the

(19). of the

A total

3 preparation. and

one

(#5)

side

of

that

peak

the

sample

appearing the

fatty

of

six

as

largest

a

peaks slight

peak

(#6).

#5 was due to oleic was

fully

acid

resolved

on

a

to mass spectrometry.

acid

data

was

washing

content Peaks

3.

of the

phospholipids

1 and

2 were

however,

peak

time

as myristic

same retention

time

and

an earlier

spectrometry,

the

3 monomer with

time

band

1.

after

identity

of

figure

remaining

suggested

fatty

of

consistent

and subjected

the

band

point

are

alone

its

time

band

band

retention

column

chromotograph

per

gas chromatogram

analysis

I presents integral

per

purified

shorter

to

the

values

standing

time

Table

Since

the

retention

methylester,

16 hour

ammonium bromide

five

the

the

lipids

These

a typical

from with

shoulder

at

phospholipids

dodecyltrimethyl

Figure

with

eluted

7 residual

column.

retained

detergent

are

3 protein,

contained

2-7

acids

gas chromatogram of the methyl esterified fatty acids of which copurify with the integral domain of the human

all 1015

other

peaks

copurified too

2 migrated acid yielded

weak in

methyl the

to the

be gas

ester.

same peak

BIOCHEMICAL

Vol. 159, No. 3, 1989 Fatty

TABLE I.

acid

composition of membrane-spanning

Peak #

'number

assignment total

14:o

<1

3

16:0

6 +

0.4

4

18:2

22 *

9.7

5

18:l

2 2

0.5

6

18:O

mass

acids

acid

total

However,

the of

was present

detergent

small the

to

associated

phospholipids

the

(18:2) 3).

fatty

acids,

phospholipid

these

less

1% of the

than

nearly

abundance

in

all Based

of the

content

the bulk

lipid

enriched co-isolate

in

-in

68% of

(22%), common

to comprise with

difficult.

3,

our

data

the

the

in oleic

(18:l)

other

major

erythrocyte

were

not

known

a

the

1).

of

In

band

(18:0) (16:0)

3 and

acids

fatty

tightly

undetectable

5-7

these

stearic

membrane in

sensitivities the

(Fig.

lipids, in

removed

residual

and palmitic

detected

that

that

bulk

enriched

acid

easily

displacement

of be

small

component.

are

show

detergent to

lipids

2%

3 lipids.

Myristic

as was the unidentified

by

fatty

only

band

combination

2 the

copurified

acid most

associated

of membrane

on the

bound

which

(
suggest

total

fivefold

phospholipids

quantitation

found

(20:4),

observations

A comparison its

acid

its

and depleted

fraction.

employed,

tightly

composition

were

acid

the

stearic

this

acid

Furthermore,

arachodonic

was estimated

at

of these

next

acid

from

band

resist

acid

the

of peak

acid

linoleic

acid,

majority

of

strongly

comparison

(Figure

vast

identification

fatty

prominent,

Oleic

amounts

the

Stearic

abundant

linoleic

rendered

extraction

phospholipids

correct.

be

most

of

peak

Although

we assume

derived

superposition in very

data,

at 6.0%.

acids

deviations

the most

next

bonds

to

After

acid

this

Discussion.

e.g.

ester

the

fatty

magnitude

linoleic

spectral

three-fold.

was palmitic the

to standard

the

68 k 11.3

number of double

was by far

exceeding

lipids,

by

2

methyl

approximately of

‘3

correspond

with

Mole %'

unidentified

acid

fatty

copurify

1

as the

as myristic

the lipids which domain of band 3

Acyl Chain type'

of carbons:

bf values

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acids,

associated the

techniques

lipids

represent

population. of stearic

acid

in the bound

phase

(Figure

3) reveals

the

former

fraction.

with

band 1016

3 constitute

that

population stearic

Whereas only

-l/30

with acid the

of the

is 5-7

total

BIOCHEMICAL

Vol. 159, No. 3, 1989

m

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Red

Cell

[7

Band

3

I

7060. 50. 40.

mol %

30.

n

16:O

IX:2

IX:1

Acyl

Chain

I x:0

IO:4

type

Fieure 3. Composition of the major (>5% for the red cell; >l% of the band 3) fatty acids of the bulk red cell membrane lipids (solid bars) and of the lipihs which copurify with the integral domain-of band 3 (shaded bars). The numbers on the x-axis indicate the number of carbons followed by the number of double bonds in each acyl chain. The data for the red cell was taken from

(20).

membrane

lipid,

the

acid.

Assuming

same population

phosphorylcholine estimated

that

bound

headgroups

phosphorylethanolamine

nearly

3/4

of

and

the

of the

species

(11,

the

cell's are

13),

cell's

total

it

phosphatidylcholine

is

exclusively

can

stearic

stearic

be

further

acid-containing

concentrated

in

this

'(20).

tightly

of long,

associating

rule

with

governing

highest

many

affinity

stabilizing of

band an

band

that

of the

series

stability

cholesterol

of fatty

band

acids

acids

are

two

predominantly

has more

previous

thick by the

content

of

the

native

revealed

the

stabilizing

with

the

aforementioned

strongly

stabilizing

the

data

been

reported

on the

acyl

1017

in

that

a

varied zwitterionic

contained

carbon

phospholipid

degree

of

acids

or

bilayer

studies with

on

the

exogenous

longer, more saturated unsaturated ones (21). that

band

zwitterionic composition

chain

important

fatty

supplemented

reported

of

long the

Analogous i.e. or

at

comprised

increasing

.18

the

calorimetric

was especially

influence chain

with effective

the

stable

reconstituted

same result, than shorter 13)

lipids

unstated

systematically

which fact

population

most

of of

bilayer

membranes

(11,

the are

protein.

ghost

lipid

bilayer

containing

transport

studies

with

most a

lipids

the

3 in

agreement have

of

stabilized

that

phosphatidylcholine)

was emphasized

a

significantly

3 was

the

to an as yet

protein

a series

(i.e., an unusually

in

examination

into

band

or That

3 stability

elevating

fatty

earlier

3 reconstituted

(10).

saturation

Although

interactions: An

phosphatidylethanolamine

chains conformity

membrane

environment

acids

acyl

reflect

may

integral

revealed

phospholipid fatty

3

protein.

band

phospholipids

saturated

protein-lipid

for

the

properties

free

of

the

The preponderance

for

-l/6

and

phosphatidylethanolamine fraction

contains

3 co-isolates

phospholipids of

these of

two

these

(in

lipids), high

affinity

no

Vol. 159, No. 3, 1989 lipids.

In

band

3 for

this

study

long,

enrichment

of

we have

saturatured

stearic

This

observation

lipids

which

active,

BIOCHEMICAL

acid

in

13)

the

band

are

that

chains

the is

be

which

of

by the

tightly

the

stable

most

lipids.

hypothesis

(and

avidly

of

selective

associating

with

more

associate

preference

satisfied

consistent

3 structurally

those

thermodynamic

also

population

therefore,

render

ref.

shown

acyl

would,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

catalytically

with

the

protein

h

vivo -. While

only

to resist

S-7 phospholipids

detergent

interactions

NMR and

adjacent (1,

The

relatively

freely

size

of

integral -40

bilayer lipids

class

perturbed

of

is

interactions

detected

in

as a result

of

mainly

the

protein

the and

phospholipids to

perfectly

matched

boundary

197

and

from

240

the

cross

(lo), in

mismatch domain

685

band will

3 be

compositions to

its

Band

withdraws regions

learn

phospholipid

how

proteins from

of

in

this

the

the

number

section

membrane's

manner

lower

which

This

cross

varies

limit

expected has

of to

a very

(27).

the

has It

the

same protein long

1018

likely size

to

as the

form short

removes

respectively,

a very

seems

large

contribute two

a

dimyristoylphosphatidylcholine

hydrophobic

dimyristoylphosphatidylcholines

these

far into environment.

weaker

transition,

core

3

composition

phospholipids

in

the

3, which

interesting, of

band

behavior yolk egg

probably

phase

while

and

must

acid

phosphatidylcholines,

(26).

transition

of

per

of an

fatty

to

lipids the

the hydrophobic

to

transition,

phase

60

A third,

and

42

distearoyl

according

to

into

glycophorin, only

nonpolar of

It the

populations influence

its in

perturbation. evaluate

and

22

perturbed

correspond

melting

varies properties

hydrocarbon

lipids

example,

which

perturbs

crystal

mismatch,

removes

transition

section

spanning

lipid

of the

phospholipids layer

the

between

of

often

motional

removal

liquid the

may

For

dipalmitoyl

same

participation large

it

segment the

of

of

system,

layer.

from

to

number

magnitude

membrane-spanning molecules

The

the

gel

classes techniques.

and membrane-spanning

the

3

to as a boundary

transporter

lipids

band

lipids",

from

unknown.

of a mismatch width

of

Unfortunately,

by

the

population

reconstituted

bilayer

avid

by our

probably

the

anion

currently

normal

(26).

according the

(25). is

calorimetrically

occurs

of

the

less

for

explored

ranges

when

between

participation

lipid,

generally

phospholipids

of

other

not

"boundary

the bulk

that

affinity

referred

these

NMR analysis

phosphatidylcholine these

with

indicates

such

of

and

(24).

lipids

clearly

were

sometimes

number

protein

protein

associated of

the

sufficient

a disordered

surface,

23).

are

which

reveal

a protein's

22,

exchange the

EPR often

to

4,

there

extraction,

protein-phospholipid Thus,

may exhibit

the

methods less

bulk

avidly lipid

from that

of

the

magnitude become

both of

this

available,

interacting phase

the

membrane-

a protein

to lipid can

Vol.

159,

No.

3, 1989

AcknowledPment.

BIOCHEMICAL

Supported

AND

by NIH grant

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

GM24417.

References 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. la.

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