Differences in composition and fluidity of intestinal microvillus membrane vesicles prepared by different methods

Vol. 170, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATiONS

July 31, 1990

Pages 433-440

DIFFERENCES

IN COMPOSITION

AND FLUIDITY

MEMBRANE VESICLES David

Received

June

PREPARED BY DIFFERENT

J. Bjorkman

VAMC and University

OF INTESTINAL

of Utah

and Earl

Medical

J.

MICROVILLUS

METHODS

Brigham

Center,

Salt

Lake

City,

Utah

9, 1990

Multiple methods have been developed to isolate the intestinal microvillus membrane and facilitate the study of its composition and function. Variations in membrane composition and fluidity may result from different preparative techniques. This study shows that the use of MgClz and/or KSCN in vesicle preparation alters phospholipid and protein composition of the membrane compared to CaCl, precipitation. The use of MgCl, in membrane preparation increased phosphatidylethanolamine and decreased phosphatidylinositol content. The use of KSCN in membrane preparation decreased the protein content. The structural changes seen with the use MgClz alone are accompanied by an increase in both static and dynamic membrane fluidity. These results suggest that different methods of membrane vesicle preparation affect membrane phospholipid and protein content as well as membrane fluidity. al990 Academic Press, Inc.

Intestinal

epithelial

and basolateral of specific Membrane

function techniques

commonly

apical

10]

depends

and separated as if

were

density there

may vary

enzymes

salts but

electron

change

in does

membrane

the

the

lipid

ABBREVIATIONS MVM, Microvillus 12-AS, DL-12-(q-anthroyl)stearic

vesicle

has

shown

the

or protein which

is

that

the

membrane; acid.

by

of

[11,12].

particle

suggesting

that

[12,15].

of membrane

vesicles

complex

lipid-protein

may

1,3,5-hexatriene;

OOO6-291x/90 433

MVM

Freeze

intramembrane

1,6-diphenyl

been

The use

enrichment

and distribution

composition

DPH,

have

and function and phospholipid

technique,

content determined

methods

structure

the

Most

Ca++ or Mg'+ [8-

proteins

preparative

protein

components. [2-123.

[5,6,10-12,141.

increases

cytoskeletal

also

with

of its

structure

technique

apical the site

absorption.

membranes cations

membrane

preparation

in membrane in

that

defined (MVM) is

nutrient

Different

to study

so by extracting

fluidity,

these

by divalent

preparative

well

relationships

centrifugation.

membrane

vary

to normal

to isolate

however,

microscopy

differences

Alterations

structural

are precipitated

with

with membrane

critical

interchangeable,

and distribution are

the

Evidenceexists,

thiocyanate

fracture

upon

polarized

microvillus

systems

by differential

they

composition marker

are highly

The apical

have been developed membranes

[2,3,5,12.13]. of

[l].

enzyme and transport

Numerous

used,

cells

domains

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

170,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

interactions. Changes in membrane fluidity membrane function [16]. The present study composition prepared

and demonstrates by different

differences

RESEARCH

COMMUNICATIONS

may have significant identifies changes

in membrane fluidity

effects on in membrane

in MVM vesicles

methods. MATERIALS AND METHODS

ANIMALS Adult male Sprague-Dawley rats weighing 225 to 300 grams (Bantin and Animals were fasted overnight Kingman, Fremont CA) were used in all studies. and anesthetized with intraperitoneal sodium pentobarbital. MICROVILLUS MEMBRANEVESICLES Microvillus membrane vesicles were prepared at 4. C by four different Two groups were prepared by standard previously described methods [8-12,173. divalent cation precipitation and two groups using additional thiocyanate The method using CaCl, with differential centrifugation was modified treatment. Alternatively, MgC& from the method of Schmitz and colleagues [8]. Both CaC12 and MgC& precipitation was used as described by Hauser [lo]. precipitated vesicles were additionally purified using KSCN by the method of Hopfer [11]. VESICLE ENRICHMENT was evaluated for membrane purity by Each preparation of vesicles determining the enrichment of the apical membrane enzymes sucrase and alkaline Protein was determined by the method of Bradford [18] using a phosphatase. Sucrase activity was measured by the commercial kit (Bio-Rad, Richmond, CA). method of Messer and Dahlqvist [19] as modified by Grand et al. [20]. Alkaline phosphatase activity was determined by the method of Bessey et al. [21]. Na+/K+ATPase activity was assessed by the method of Scharschmidt et al. [22]. Mean enrichment of sucrase was 16.8-fold for CaC&. 18.5 for MgCl,, 34.9 Alkaline for CaCl, with KSCN and 28.9 for MgCl, with KSCN prepared vesicles. phosphatase enrichment was 10.6 for CaC12. 13.6 for the MgCl,, 20.0 for the CaCl, with KSCN and 22.3 for the MgCl, with KSCN preparations. Basolateral membrane contamination was minimal in all groups, as determined by decreased Na'/K'ATPase activity. Electron microscopic evaluation of vesicle pellets from each group was similar to that published previously [12]. There was no microscopic evidence of contaminating membrane populations, LIPID ANALYSIS Membrane lipids were extracted from MVM vesicles by chloroform:methanol extraction [23]. Cholesterol content was determined enzymatically [24] using a commercial kit (Sigma Chemical Co., St. Louis, MO). Phospholipid composition was determined by two-dimensional thin layer chromatography of plates coated with silicagelH [25]. Chloroform-methanol-28% ammoniumhydroxide (65:25:5) was used in first the system and chloroform-acetone-methanol-acetic acid-water (3:4:1:1:0.5) was used in the second system. Individual phospholipid content was determined from gel scrapings using a modification of the method of Bartlett

L.261. MEMBRANEFLUIDITY MEASUREMENT Static and dynamic fluidity of MVM vesicles was estimated by measuring steady state fluorescence anisotropy using the method of Schachter and Shinitxky 1,3,5-hexatriene (DPH) and DL-12-(9-anthroyl) stearic acid C27]. 1,6-diphenyl (12-AS) were used as probes. Steady state fluorescence anisotropy (r) was measured immediately at 24. C using a SLM 4800 fluorometer (SLM Instruments, Inc., Urbana IL) with an excitation wavelength of 360 nm for DMH and 365 nm for 12-AS. Emission above 397 nm was measured using cut-off filters. Fluorescence anisotropy was calculated according to the equation r=(I I )/ (I +21 ) [28]. Corrections for light scattering and ambient fluorescence were made using vesicle suspensions without probe and by pelleting membrane

434

Vol.

170, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from the medium. Excited state lifetimes were determined for each probe using both phase shift and frequency modulation methods at 6 MHz and 18 MHz. Lifetime measurements did not vary significantly between vesicle preparations for either probe, suggesting that differences in anisotropy were not due to differenti.al probe quenching or other probe related factors.

vesicles

STATISTICAL ANALYSIS Statistical significance of all data was determined using a two-tailed Student's t-test for independent samples. RESULTS PHOSPHOLIPIDANDCHOLESTEROL COMPOSITION The phospholipid composition profile is shown in figure 1. MgCl;, precipitation with or without KSCN treatment significantly increasedvesiclemembranephosphatidylethanolamine with CaCl,, precipitation. Vesicle preparation

The use of (p
comparedtovesiclesprepared with MgCl* alone decreased

membranephosphatidylinositol content compared to other groups (~(0.05). There was a slight increase in lysophosphatidylcholine in the vesicles prepared with CaClz alone,

but

lysophospholipids vesicle

group.

this

did

not

achieve

statistical

comprised less than 2% of the total Treatment with KSCNdid not Alter

significance. lipid

Other

phosphorus of any

the phospholipid

composition

of the membranevesicles.

PC

cl CaCl, Figure

1.

LPC

SPHINGO

PE

q NCb

PS

q Ca/KSCN

PI

FFA

LPL

q Mg/KSCN

Phospholipid composition determined by two dimensional thin layer Values are chromatography of MVM prepared by different methods. expressed as the percent of total lipid phosphorus f the standard PC=phosphatidylcholine. error of the mean (n=ll for all values). LPC=lysophosphatidylcholine, SPH=sphingomyelin, PE=phosphatidylethanolamine. PS=phosphatidylserine, PI=phosphatidylinositol. FFA= free fatty acids, LPL= less polar lipids. Lysophosphatidylethanolamine represented less than 2% of each group. *PE was higher in each magnesium treated group (pcO.01). **PI in the MgCl, group was lower than all other groups (pcO.05).

435

Vol.

170,

No.

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

40 x

30

T

I

10

CaC1,

q )CHOLESTEROL:PROTEIN Figure

CaCl,

Treatment

with

prepared

vesicles

(figure

KSCN increased

This

group

f

0.016

but lipid

not

groups

(17.95

* 3.29

CaCl,

precipitation

ratio

alone value

(7.57

and 15.87 * 0.95)

(10.8

* 1.89)

0.277

with

was not

0.030 in

the with

to the CaCl,

MgC12 (p>O.O5).

was also

(p
f

in

when treated

when compared

* 3.52

that

to

(ug/mg)

had an increase

* 0.038

significant prepared

SRM)

also

to 0.305

the vesicles calcium

ratio

f

@Cl,

* 0.029

phosphorus:protein with

(mean

with

was statistically

(p
increased

in KSCN treated

with

magnesium)

MgCl,

precipitation

statistically

compared

to

resulted different

from

group.

The cholesterol:total betweenvesicle with

PHOSPHORUS:PAOTEIN

cholesterol:protein

prepared

from 0.216

difference

in an intermediate

the

0.157

Vesicles ratio

The total

any other

from

2).

cholesterol:protein KSCN.

q

Mg/KSCN

TOTAL LIPID

Cholesterol:protein (ug/mg x 10) and total lipid phosphorus:protein ratio for MVMprepared by different techniques. Values are expressed as the ratio f the standard error of the mean (n=ll for all values). 'Increased the cholesterol:protein ratio compared to CaCl, treatment alone (p
2.

(p
CalKSCN

MU,

respect

lipid

groups, to total

be responsible

for

our

noted

previously

phosphorus

ratio

suggestingthatcholesterol phospholipids

the other

and that

ratio

extraction

did

not

vary

composition

changes.

differences These

of cytoskeletal

in protein

findings

proteins

significantly

remains are

unchanged content

consistent

may with

by KSCN [12].

FLUORESCENCE ANISOTROPY Fluorescence lower

in

anisotropy.

membrane

groups

(p
dynamic

membrane

in

determinedbyboth

vesicles all fluidity

prepared

cases, are

table

using 1).

increased 436

DPH and12-AS MgCl 2 alone

This

indicates

in this

group.

was significantly

compared that

both

to

all static

other and

Vol. 170, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1

FLUORESCENCEANISOTROPY OF MICROVILLUS MEMBRANE PREPARATIONS

Preparative

Fluorescence

Group

DPH

Anisotropy

(r) Q-AS

CaC12

0.276

f 0.001

0.135

f 0.002

MgCL

0.267

t 0.002~

0.129

* 0.002,

CaCl, + KSCN

0.277

i 0.002

0.139

f 0.002

MgCla + KSCN

0.278

f 0.003

0.139

f 0.002

Fluorescence anisotropy (r) determined by steady state fluorescence polarization using DPH and 12-AS as probes in microvillus membrane vesicles prepared by different methods. Values are r value f SFM. * p
DISCUSSION These the

data

suggest

prepar.ative

with

an increased

vesicles. noted

These

role

thiocyanate

are

extracts

are

cases, consistent

studies True

our

previous

changes

for

[Zq].

remained

profile

of intramembrane

MgCl,

seen with

by the extraction

showing

the protein

with

ratios

ratio results

who also

in view of the broad

messenger

can be accounted

and distribution

membrane within

difficult

to achieve membrane

the

lipid

different

fluorescence

polarization

types

A variety

"fluidity". been

the

that

of

unchanged.

KSCN treatment

on SDS-PAGE analysis

particles

used

fluidity

to estimate of

freedom

An exact

bilayer.

because

have overall

describes

"fluidity"

limitations, estimating

with

prepared

Mg+' precipitation

decreased

significance

cholesterol:phospholipid

vesicles

and colleagues, is

with

seen in freeze

[12].

molecules overall

membrane

with

as a second

vary

to calcium

and phospholipid:protein

proteins,

the number

fracture

of Aubry content

of the vesicles

cytoskeletal

and alters

to those

may be of particular

the

provides compared

phosphatidylethanolamine

cholesterol:protein

.In all data

precipitation

phosphatidylinositol This

and composition

content

similar

membrane

treatment

protein.

structure

of phosphotidylinositol

The higher

These

in

alone.

physiologic

Magnesium

results

Additionally,

precipitation

MVM vesicle

phosphatidylethanolamine

an i,ncrease

t-141.

that

technique.

of molecular of

techniques,

membrane

membrane

probes

[14,27,28,30-331. 437

of

motion

determination

order has been Because

of of

motion each

is

contribute

to

having

C27.281. widely of

membrane

fluidity

their

its

Steady

own state

accepted rotational

for

Vol.

170,

No.

2, 1990

characteristics AS are

and localization

often

fluidity

used

to

static is

and dynamic to

simultanteous phosphatidylinositol change

in

membrane

presence

or absence

exposure

in

vivo

in

one

it A

factor.

and decrease unlikely that

is

change

acid

in

length

in the

phospholipid and saturation,

in

be noted

that

vesicles

studies

irreversibly

a variety

and fatty

were

technique addition

calcium

to calcium

exposure

these

the

MVM by at

composition

calcium

through

that

in vitro

vivo

precipitation that

by

intestinal

in phospholipid

suggest

of membrane

have shown of

of vesicles

while

same MgClz

together

may be affected

fluidity

exposure

[38],

should

the

a

fluidity the

an alteration

by

[28,35-373,

any

It

and colleagues

Direct

It

to

in fatty

vesicle

Brasitus

fluidity

through

the fluidity

solely

may decrease

mechanisms.

studies

to

Because

methods.

of influences

fluidity

a higher

changes.

of calcium.

prepared

in

due

membrane

[38,x9].

These

affect

observed

decrease

vesicles

membrane

have

by other

a variety

increase

is

or in vitro

fluidity

study.

of

precipitation

prepared

of

such as changes

that

two different

saturation

DPH and 12-

components

in membrane phosphatidylethanolaine seen with Mg+' precipitation.

the

exists

MgCl,

vesicles

this

factors,

in

may reversibly

COMMUNICATIONS

of the membrane

and dynamic with

result

fluidity

Other

decreases

the

attribute

a role

RESEARCH

depths

static

prepared than

is

is

Evidence

least

the

fluidity

increase

composition. may play

acid

estimate

fluidity

difficult

BIOPHYSICAL

in different

MVM vesicles

membrane

AND

[28,34].

respectively Intestinal

net

BIOCHEMICAL

performed

used or

in

this

depletion

of direct

may

and indirect

mechanisms. It of

has been established

membrane

have

that

the

vesicle

regarding is likely system.

vesicles

that

are

fracture

analysis

activates

membrane

Neither

this

increase

in

study

groups

that

all

We have [12].

differ

from

MgC12

precipitation

CaCl,

phosphatidylinositol

although

content

intestinal

Aubry

alter

Meddings

the

in

and

colleagues axis

between

composition

this

a variety

crypt-villus

of the relationship study

introduce that

is

membrane

and

may have at least

CaCl,

fluidity important

changes

on freeze that [14]

provides

tissue fracture CaCl,

lysophospholipid

and colleagues

some artifact

preparation

intestinal

have suggested

and increases with

on freeze with

content noted

MgClz

precipitation [lo].

a significant

the use of calcium.

the mechanisms

increases

the

to intact

have noted

of

preparations

example,

shown

and colleagues that

For

methods

similar

phospholipases

may directly

functions.

previously

Others

nor

noted

membrane

structurally

fluidity

function,

preparative

lysophospholipids

In summary,

Because and

various

[ 153. Hauser

preparation

along

[ho]. fluidity

between

implications It

transport

fluidity

structure,

differences

into

glucose

to membrane

composition,

membrane

[16,30.31,38,39].

functions

shown

related

that

in membrane

are not

clear,

composition,

structure

phosphatidylethanolaine and increases

438

static

prepared

MgCl>

and fluidity.

content, and dynamic

vesicles

membrane

decreases fluidity.

Vol.

The

170,

No.

use

of

differences

2, 1990

BIOCHEMICAL

KSCN alters should

membrane

be considered

AND

BIOPHYSICAL

structure in

studies

and using

RESEARCH

protein

COMMUNICATIONS

composition.

These

MVM vesicles.

ACKNOWLEDGMENTS This project and a Biomedical

was supported by the Veterans Administration Research Support Grant from the University

Research of Utah.

Service

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