BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vo1.186, No. 3,1992

Pages 1294-1298

August 14, 1992

E F F E C T OF GLYCOLIPIDS ON THE PHASE BEHAVIOR AND DYNAMIC PROPERTIES OF PHOSPHOLIPID LIPOSOMES

Jian-Wen

Institute

Received

Chen*,

Ke Shi, Langping

of Biophysics,

June 23,

Academia

Zhang and Fen Huang

Sinica,

Beijing

I00101 China

1992

S u m m a r y : T h e g l y c o l i p i d s of A c h o l e p l a s m a laidlawii AIH089 membranes were i d e n t i f i e d a n d purified. The effect of m o n o g l u c o s y l d i a c y l g l y c e r o l ( M G D G ) and diglucosyldiacylglycerol(DGDG) on the thermotropic b e h a v i o r of m u l t i l a m e l l a r v e s i c l e s of d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e ( D P P C ) , dipalmitoylphosphatidylglycerol(DPPG) has been investigated by high sensitivity differential scanning calorimetry. T h e m a i n t r a n s i t i o n peaks were b r o a d e n e d , t h e e n t h a l p i e s w e r e decreased. DGDG caused the decrease in the transition temperatures of DPPC, DPPG liposomes by 3.08°C, 4.18°C, respectively. MGDG did not cause the a l t e r a t i o n of t h e t r a n s i t i o n t e m p e r a t u r e of D P P C l i p o s o m e s but c a u s e d t h e d e c r e a s e of t h e t r a n s i t i o n t e m p e r a t u r e s of DPPG l i p o s o m e s by 2.20°C. E S R e x p e r i m e n t s i n d i c a t e that M G D G d e c r e a s e d the r o t a t i o n a l c o r r e l a t i o n t i m e of DPPC and DPPG liposomes. © 1992AcademicPress, mnc.

Glycolipid

is an important

microorganisms. glycosphingolipids Mycoplasma membrane are

the

total

Both

dominant

hexagonal

interaction we r e p o r t

found

divided

into

of

acid

in animals, two

which occur

lipids

have

usually

It has b e e n

accounting

reported

that

the DGDG/MGDG composition

of glycolipids

with

of M G D G

ratio

(7,8).

phospholipid and DGDG

and dynamic properties

MATERIALS

50%

by

liposomes.

from A . l a i d l a w i i of DPPC and DPPG

is

weight

known

AIH089

of

the

exhibited

structure

In the

are

and DGDG

to m a n i p u l a t i o n s

little

and

modulate

MGDG

liposomes

a bilayer

responds Very

to

the glycolipids (5,6).

for

and

classes:

bacteria

potential

that M G D G

exhibited

plants,

distinct

in plants,

the

It is well k n o w n

while DGDG liposomes

the effects

the t h e r m o t r o p i c

(4).

glycolipids,

laidlawii,

fatty

of lipid

be

lipid of the A. laidlawii m e m b r a n e s

lipids.

II phase,

Acholeplasma membrane

classes

properties

constituent

membrane

can

and glycoglycerolipids,

(1,2,3).

physical

the m a j o r

class

Glycolipids

(3). of

about

present

a In

the the

study

membranes

on

liposomes.

AND METHODS

M a t e r i a l s : A . l a i d l a w i i A I H 0 8 9 w a s s u p p l i e d b y t h e I n s t i t u t e of A n i m a l H u s b a n d r y and V e t e r i n a r y Medicine, Jiangsu A c a d e m y of A g r i c u l t u r a l S c i e n c e . DPPC a n d D P P G w e r e p u r c h a s e d from Sigma and s h o w e d a s i n g l e spot b y t h i n layer chromatography. The spin label probes, 5 - ( N - o x y l ) 4 - 4 - d i m e t h y l o x a z o l i n e

*

To w h o m correspondence

should be addressed.

0006-291X/92 $4.00 Copyr~ht © 1992byAcademic Press, Inc. Allr~h~ofreproduction in anyform reserved.

1294

Vol. 186, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(5NS) a n d 1 6 - ( N - o x y l ) - 4 - 4 - d i m e t h y l o x a z o l i d i n e ( 1 6 N S ) derivatives a c i d o b t a i n e d f r o m SYVA. O t h e r r e a g e n t s were of a n a l y t i c a l grade. C e l l qrowth:

A. l a i d l a w i i

strain A I H 0 8 9

was c u l t u r e d

of

as d e s c r i b e d

stearic

before(9).

Isolation and purification of q l y c o l i p i d s ( 1 0 ) : A.laidlawii AIH089 cell w e r e h a r v e s t e d by c e n t r i f u g a t i o n and lipids were e x t r a c t e d f r o m t h e c e l l s w i t h water-saturated n-butanol. The solvent was removed a n d t h e lipids w e r e f u r t h e r extracted by partition in biphasic chloroform-methanol-water system(8:4:3 v/v). Lipids were collected and the solvent was removed by a stream of n i t r o g e n . T h e l i p i d s w e r e t h e n a p p l i e d to a silicic a c i d column. E l u t i o n w a s commenced with chloroform-acetone (1:4 v / v ) a n d t h e s o l v e n t changed to acetone, MGDG and DGDG were collected separately. Glycolipids were further purified by thin-layer chromatography on s i l i c a g e l G w i t h t h e d e v e l o p i n g solvent chloroform-acetone-acetic a c i d ( 6 : 6 : l v/v). M G D G a n d D G D G w e r e e s t a b l i s h e d w i t h Rf value, fatty acid c o m p o s i t i o n of M G D G and D G D G w a s a n a l y z e d by gas c h r o m a t o g r a p h y a c c o r d i n g to the p r e v i o u s p r o c e d u r e ( l l ) . P r e p a r a t i o n of liposomes: M u l t i l a m e l l a r l i p o s o m e s w e r e p r e p a r e d by d i s p e r s ing at c o n c e n t r a t i o n 1-2 m g of lipids p e r ml of T r i s - H C l b u f f e r , pH 7.4, in a v o r t e x for 2 m i n (above t h e lipid t r a n s i t i o n t e m p e r a t u r e ) . L i p o s o m e s u s e d in E S R e x p e r i m e n t s w e r e p r e p a r e d a c c o r d i n g to t h e p r e v i o u s study(12). DSC measurement: Calorimetric measurements were performed with a Microcal MC-2 d i f f e r e n t i a l s c a n n i n g calorimeter. T h e c o n c e n t r a t i o n of t h e lipid w a s 1 mg/ml. The c a l o r i m e t r i c scans were p e r f o r m e d at a s c a n n i n g r a t e of 45°C/h. S p i n label and E S R m e a s u r e m e n t : p h o s p h o l i p i d l i p o s o m e s (20mg/ml) c o n t a i n e d d i f f e r e n t c o n c e n t r a £ i o n s of M G D G or D G D G at a m o l a r ratio. T h e c o n c e n t r a t i o n of 5NS or 1 6 N S w a s i m g / m l . E S R s p e c t r a w e r e r e c o r d e d w i t h B r u k e r E R 2 0 0 D - S R spectrometer. The spectrometer was equipped with a variable temperature c o n t r o l l e r and a d i g i t a l thermometer. T h e m i c r o w a v e p o w e r w a s 19.7 mW, X-band, m o d u l a t i o n f r e q u e n c y w a s i00 KHz w i t h an a m p l i t u d e of 1 G. T h e field s w e e p was 200 G and t i m e c o n s t a n t 0.128. The o r d e r p a r a m e t e r was c a l c u l a t e d a c c o r d i n g to Hubbel and McConnell(13) from the spectra of 5 N S . The rotational c o r r e l a t i o n t i m e w a s c a l c u l a t e d a c c o r d i n g to K i v e l s o n ( 1 4 ) f r o m t h e s p e c t r a of 16NS.

RESULTS The AIH089 acids

fatty

were

very

Typical or DGDG

DPPC

centered

enthalpy

at

shown 42°C,

of D P P C

in Fig.l. with

is 7.42

that

of M G D G

in T a b l e

and D G D G

i.

The

isolated

saturated

and

from

No s i g n i f i c a n t

liposomes

A sharp When

change

detected

DPPC

transition

peak

in t h e

alone

and

transition

pretransition

Kcal/mol.

the main

at

liposomes broadens

phase

in t h e

was

fatty

12:0 6.93 5.72

14:0 16.67 14.45

16:0 30.80 33.82

35°C.

The

containing

1295

with

18:1 19.73 21.15

i0 mol%

temperature

18:2 9.73 9.59

of pure

transition

and pretransition

transition

18:0 16.13 15.27

presence

obtained

Table i. Acyl-Chain Composition (%) in MGDG and DGDG Isolated from A.laidlawii AIH089 acyl chain MGDG DGDG

A.laidlawii

unsaturated

in b o t h of g l y c o l i p i d s .

thermograms

were

indicate

showed

similar

of D P P C

disappears.

compositions

were

DSC

MGDG

MGDG

acid

membranes

AND D I S C U S S I O N

of

peak

of D P P C

V o l . 1 8 6 , N o . 3, 1 9 9 2

B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

A

o

o

_o

B

B

I

zo

i

2b

12

Q

|

i

28

36

|

44

20

TEMPERATURE °C

25 '

3b 3% 40 TEMPERATURE °C

Q

45

Figure I. DSC thermograms for DPPC liposomes. (A) Containing 0 (a), I0 (b), 20 (c), 30 (d) mol% MGDG~ (B) containing 0 (a), i0 (b), 20 (c), 30 (d) mol% DGDG. Figure 2. DSC thermograms for DPPG liposomes. (A) Containing 0 (a), i0 (b), 20 (c), 30 (d) mol% MGDG; (B) containing 0 (a), i0 (b), 20 (c), 30 (d) mol% DGDG.

c a n be o b s e r v e d , from

7.42

increasing of

DGDG,

I°C

DGDG

and

of D P P G

further

studied.

transition

causes

peak,

were

molar

lower

ratio

of

curves

to 5.2 K c a l / m o l

and DGDG have stronger

of D P P G

for DPPG.

the

of D P P G

in the

decreased

two

4.18.

1296

high

main

for DPPG.

DPPG

by

At

from

by

7.42

containing

concentration

The

became

enthalpies

In s a m e c o n d i t i o n ,

bilayer The

on t h e behavior

transition

by 2 . 2 0 ° C .

organization.

enthalpies

F r o m t h e s e data,

interaction with DPPG

DPPC.

lower

of D G D G

glycolipids

liposomes

in Fig.2.

to 5.4 K c a l / m o l

DPPG

a

temperatures

decreased

on t h e t h e r m o t r o p i c

30:70),

temperatures

alterations

also

of t h o s e

showed of

to

respectively(Fig.iB).

and DGDG

The D S C

DPPC

by

In case

concentration

of t r a n s i t i o n

of D P P C

decreased

(Fig.iA).

the

as t h e

are

respectively,

and 30 m o l %

mol%

effect

of M G D G

of M G D G

caused

temperatures

from 8.69 Kcal/mol both MGDG

to

the

f r o m 8.69 K c a l / m o l

significantly

I0

and 5.2 K c a l / m o l ,

(MGDG:phospholipid

also decreased DGDG

of

of D P P C

Kcal/mol,

M G D G to 20 m o l %

of t r a n s i t i o n

the effect

was

shifted

5.4

and 30 mol% w i t h a d e c r e a s e

concentrations

and

and

of p r e t r a n s i t i o n

to e l u c i d a t e

liposomes

of

enthalpies

phospholipid,

MGDG

broader

The

of t h e t r a n s i t i o n

Kcal/mol

a concentration

to 5.7 K c a l / m o l

different of

at

to i0 m o l %

In o r d e r acidic

enthalpies

5.6

and v a n i s h i n g

3°C.

Kcal/mol

the

to

the c o n c e n t r a t i o n

temperature increases

while

Kcal/mol

The

decreased

it is c l e a r t h a t

liposomes

t h a n t h a t of

Vol. 186, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 2. Effect of MGDG and DGDG on Order and Mobility of DPPC and DPPG Liposomes# Sample

Order parameter

DPPC DPPC+MGDG DPPC+DGDG DPPG DPPG+MGDG DPPG+DGDG

Rotational correlation time x 10 -9 sec 3.46±0.07 3.13±0.05 (p

Effect of glycolipids on the phase behavior and dynamic properties of phospholipid liposomes.

The glycolipids of Acholeplasma laidlawii AIH089 membranes were identified and purified. The effect of monoglucosyldiacylglycerol (MGDG) and diglucosy...
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