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