Báo cáo khoa học về tổ chức Lateral trong mô hình lớp lipid kép Acholeplasma laidlawii chứa đầu dò pyrene nội sinh

Lateral organization in

Acholeplasma laidlawii

lipid bilayer

models containing endogenous pyrene probes

Patrik Storm

,LuLi

, Paavo Kinnunen

3,4

and A

˚ke Wieslander

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden;

Wallenberg Laboratory for

Cardiovascular Research, Go

¨teborg University, Sweden;

Department of Medical Chemistry, Institute of Biomedicine,

Helsinki University, Finland;

Memphys – Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark

In membranes of the small prokaryote Acholeplasma laid-

lawii bilayer- and nonbilayer-prone glycolipids are major

species, similar to chloroplast membranes. Enzymes of the

glucolipid pathway keep certain important packing proper-

ties of the bilayer in vivo, visualized especially as a monolayer

curvature stress (spontaneous curvature). Two key enzymes

depend in a cooperative fashion on substantial amounts of

the endogenous anionic lipid phosphatidylglycerol (PG)

for activity. The lateral organization of five unsaturated

A. laidlawii lipids was analyzed in liposome model bilayers

with the use of endogenously produced pyrene-lipid probes,

and extensive experimental designs. Of all lipids analyzed,

PG especially promoted interactions with the precursor

diacylglycerol (DAG), as revealed from pyrene excimer ratio

(Ie/Im) responses. Significant interactions were also recorded

within the major nonbilayer-prone monoglucosylDAG

(MGlcDAG) lipids. The anionic precursor phosphatidic

acid (PA) was without effects. Hence, a heterogeneous lateral

lipid organization was present in these liquid-crystalline

bilayers. The MGlcDAG synthase when binding at the PG

bilayer interface, decreased acyl chain ordering (increase of

membrane free volume) according to a bis-pyrene-lipid

probe, but the enzyme did not influence the bulk lateral lipid

organization as recorded from DAG or PG probes. It is

concluded that the concentration of the substrate DAG by

PG is beneficial for the MGlcDAG synthase, but that

binding in a proper orientation/conformation seems most

important for activity.

Keywords:Acholeplasma; chemometrics; lipid heterogeneity;

pyrene.

Acholeplasma laidlawii A-EF22 is a simple cell-wall-less

prokaryotic parasite. Its membrane lipid composition is

metabolically adjusted in response to environmental and

lipid-supply conditions. Due to this, A. laidlawii has been

used as a model system to study plasma membrane

properties and how these are maintained by the lipid

synthesizing enzymes. Membrane lipids are synthesized in

two competing pathways, both using phosphatidic acid (PA)

as a precursor, with one branch resulting in glucolipids and

the other in phosphatidylglycerol (PG) as shown in the

diagram below.

At least five enzymes constitute the glucolipid pathway.

Phosphatidic acid phosphatase (PAP) makes diacylglycerol

(DAG) from PA. 1,2-diacylglycerol-3-glucosyltransferase

Correspondence to A

˚ke Wieslander, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

Fax: + 46 8 15 36 79, Tel.: + 46 8 16 24 63, E-mail: ake@dbb.su.se

Abbreviations: bis-PyrPC, 1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphatidylcholine; bis-PyrPG, 1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-

glycero-3-phospho-rac-glycerol; CL, cardiolipin; 1,2-DOG, 1,2-dioleoylglycerol; DGlcDAG, 1,2-diacyl-3-O-[a-

-glucopyranosyl-(1ﬁ2)-O-a-

-glucopyranosyl]-sn-glycerol; MADGlcDAG, 1,2-diacyl-3-O-[a-

-glucopyranosyl-(1ﬁ2)-O-(6-O-acyl-a-

-glucopyranosyl)]-sn-glycerol;

MAMGlcDAG, 1,2-diacyl-3-O-[6-O-acyl(a-

-glucopyranosyl)]-sn-glycerol; MGlcDAG, 1,2-diacyl-3-O-(a-

-glucopyranosyl)-sn-glycerol; PA,

phosphatidic acid; PC, phosphatidylcholine; PD, pyrenedecanoic acid; PG, phosphatidylglycerol; PyrDAG, 1-palmioyl-2-pyrenedecanoyl-

glycerol; PyrPA, 1-palmioyl-2-pyrenedecanoyl phosphatidic acid; PyrPG, 1-palmioyl-2-pyrenedecanoyl-phosphatidylglycerol.

Enzymes: 1,2-diacylglycerol-3-glucosyltransferase (MGlcDAG synthase; EC 2.4.1.157); 1,2-diacylglycerol-3-a-glucose (1ﬁ2)-a-glucosyl

transferase (DGlcDAG synthase; EC 2.4.1.208).

(Received 30 November 2002, revised 22 January 2003, accepted 19 February 2003)

Eur. J. Biochem. 270, 1699–1709 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03527.x

(MGlcDAG synthase) (EC 2.4.1.157; Iabove), makes

monoglucosyl diacylglycerol (MGlcDAG) from DAG plus

UDP-Glc. 1,2-diacylglycerol-3-a-glucose (1ﬁ2)-a-glucosyl

transferase (DGlcDAG synthase) (EC 2.4.1.208), II above,

makes diglucosyl diacylglycerol (DGlcDAG) from MGlc-

DAG and UDP-Glc. Under certain circumstances, when

MGlcDAG turns bilayer-prone by saturated chains, the

more acylated and more nonbilayer-prone minor glu-

colipids, i.e. MAMGlcDAG and MADGlcDAG, are

synthesized [1]. Likewise, substantial amounts (20–30 mol/

100 mol) of the normally minor precursor 1,2-DAG may

accumulate in membranes with many saturated acyl chains [2].

It has been shown that the lipid composition is regulated

to maintain certain properties: (a) a balance between bilayer

and nonbilayer lipids (e.g. MGlcDAG/DGlcDAG) yielding

phase equilibria close to a bilayer/nonbilayer transition; (b)

a certain surface charge density through the ratio between

the glucolipids (MGlcDAG, DGlcDAG and more acylated

variants) and the charged lipids (PG and the phosphoryl-

ated glucolipids). In vivo it has been shown that the ratio

between DGlcDAG and PG is nearly constant [3]. The

regulation of these properties is sensed and performed by

the lipid synthesizing enzymes. Each enzyme acts on a lipid

substrate with a specific headgroup, but are also sensitive to

the type of acyl chains and lipid composition in the

membrane [2].

MGlcDAG synthase (I) is activated by approximately

20 mol/100 mol PG or 10 mol/100 mol cardiolipin (CL),

but is not critically dependent on the nature of the

phosphate moiety and can be activated by other negatively

charged lipids, however, not as efficiently [4–7]. The

activation by CL indicates no specificity for the PG

headgroup, but that the negatively charged phosphate is

important for the enzyme.

DGlcDAG synthase (II) is activated by PG or CL in the

same way as MGlcDAG synthase, but also by other

phosphate-containing species such as certain metabolites

and dsDNA [8]. However, PG is the strongest activator

among the naturally occurring lipids (strain A-EF22 does

not make CL). As for the MGlcDAG synthase, this process

is cooperative with respect to PG amounts and has a fairly

high Hill coefficient (4–6 for MGlcDAG synthase and 3–7

for DGlcDAG synthase) [4]. Substrate fractions of MGlc-

DAG up to five mol/100 mol raise the activity, which then

levels out, most likely due to saturation of the active site [8].

More DGlcDAG is made from MGlcDAG in membranes

with more unsaturated or longer acyl chains, increased

temperature or increased amount of cholesterol. The shift in

this lipid ratio stems from the more pronounced nonlamellar

tendency of the membrane, compensated by making more

DGlcDAG (from the nonbilayer prone MGlcDAG). This

curvature sensitivity implies a sensing mechanism of mem-

brane perturbation of nonbilayer-prone lipids. Analogous

sensing features have been proposed for CTP:phosphocho-

line cytidylyltransferase [9–11] or protein kinase C [12].

Could lipids adopting a heterogeneous lateral distribution

have a bearing on the activity of the A. laidlawii glucosyl-

transferases, in that substrates or surface charges become

locally concentrated? Mammalian plasma membranes show

transverse and lateral asymmetry. In the outer leaflet, rafts

can form by the tight packing of saturated glycosphingo-

lipids and cholesterol in a L

phase, possible to isolate

[13–16]. The biological function seems to be enrichment of

certain proteins, e.g. doubly acylated or GPI anchored in

the rafts[17–21] involved in signaling and transport over

the membrane. In the inner leaflet, lateral heterogeneity can

form with phosphatidylserine and diacylglycerol, activating

protein kinase C [22]. In this respect DAG is special in the

interspacing, dehydration and altering conformation of lipid

headgroups, as well as conferring a nonbilayer propensity

for the membrane [23].

The reason for lateral heterogeneity is preferential

interaction between headgroups (Coulombic forces, hydro-

gen bonding, divalent cations, hydration level) or acyl

chains (London forces) of certain lipids [24,25]. It is known

that acyl hydrocarbon chain mismatch can cause lateral

segregation, either by the length or the degree of saturation

[5,26–30]. Indeed, stability of raftsdemands a critical

mismatch, as POPE (1-palmitoyl-2-oleoyl-sn-glycerophos-

phatidylethanolamine) but not PDPE (1-palmitoyl-

2-docosahexanoyl-sn-glycerophosphatidylethanolamine)

mix with raft lipids [31]. In vivo lipid mixtures from

Micrococcus luteus and A. laidlawii, both containing gly-

colipids and PG, reveal interactions between the individual

lipids in monolayer experiments [32,33]. Analogous features

are also recorded for plant galactolipids. The importance of

the glycolipids for these properties are highlighted by the

lower lateral diffusion for A. laidlawii in vivo glucolipids

compared to the E. coli phospholipids [34].

To investigate whether lateral heterogeneity exists in the

fluid glucolipid-rich membrane of A. laidlawii A-EF22 as a

function of headgroup composition, liposomes were made

where composition of five different lipids (major lipids in the

membrane of A. laidlawii A-EF22), all with di-18:1c acyl

chains, was varied according to a chemometrical experi-

mental design. Pyrene-derivatives of the same lipids, inclu-

ding endogenous major glucolipids synthesized by

A. laidlawii, were used as fluorescent probes. A potential

influence on the MGlcDAG synthase, the first regulating

enzyme in the glucolipid pathway, was also investigated.

Materials and methods

Lipids and probes

MGlcDAG and DGlcDAG were prepared from A. laidla-

wii cells grown in a lipid-depleted medium supplemented

with oleic acid [35]. 1,2-dioleoylglycerol (1,2-DOG) was pur-

chased from Larodan (Malmo

¨, Sweden). Phosphatidylgly-

cerol (PG) was purchased from Avanti polar Lipids (USA).

Pyrenedecanoic (PD) acid, 1-palmitoyl-2-pyrenedecanoyl-

phosphatidylglycerol (PyrPG) and 1,2-bis-[10-(pyren-1-yl)]

decanoyl-sn-glycero-3-phosphatidylcholine (bis-PyrPC) was

purchased from Molecular Probes Inc. (Oregon, USA).

1-palmioyl-2-pyrenedecanoyl-glycerol (PyrDAG) and

1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phospho-rac-

glycerol (bis-PyrPG) and 1-palmioyl-2-pyrenedecanoyl

phosphatidic acid (PyrPA) were from KKV Bioware

(Espos, Finland).

Organism and growth conditions

A. laidlawii strain A-EF22 was grown at 30 C in a lipid-

depleted tryptose/bovine serum albumin medium [36]. The

1700 P. Storm et al.(Eur. J. Biochem. 270)FEBS 2003

fatty acids, oleic (18:1c) and palmitic (16:0) were supple-

mented from sterile ethanol stock solutions and pyrene-

decanoyl (PD) acid was supplemented from sterile

dimethyl sulfoxide stock solution. Total concentration of

fatty acids was 150 l

in the growth medium. Fatty acids

were radiolabeled with 10 lCiÆL

[

C]palmitic and

100 lCiÆL

[

H]oleic acid (Amersham Pharmacia Biotech,

Uppsala, Sweden), respectively, after four consecutive

inoculations. 2-hydroxy-propyl-b-cyclodextrin (10 m

)

was used in the medium as a carrier for the PD acid

[37]. Cell growth was monitored by absorbance and by

phase contrast light microscopy. Contamination by any

other bacteria was analyzed on standard bacteriological

agar plates.

Extraction and analysis of lipids

Cells were harvested by centrifugation, washed twice in

buffer, and frozen at )80 C. Membrane lipids were

extracted from the cell pellets using chloroform/methanol

(2 : 1, v/v).

One-dimensional thin layer chromatography (TLC) was

used to separate and characterize the different lipids in the

membrane. The TLC plates coated with silica gel 60

(Merck, Darmstadt, Germany) were developed in chloro-

form/methanol/water (80 : 25 : 4, v/v/v). [

C]-labeled

lipids were visualized with electronic autoradiography

(Packard Instant Imager). Excised gel lipid spots were

digested in Soluene-350 (Packed) for 30 min at 37 Cand

quantified by double-channel liquid scintillation counting.

To purify the pyrenyl lipids, the TLC plate was developed

first in chloroform/methanol/water (80 : 25 : 4, v/v/v) and

then in chloroform/methanol/ammonia (91 : 35 : 10, v/v/

v). Compared with a one-dimensionally developed TLC

plate of extracted lipids from medium 18:1c/PD

120 l

:30l

, the spots of pyrenyl lipids could be

separated better and become more concentrated in two

dimensions. Excised gel spots of pure pyrenyl glucolipids

(MGlcDAG and DGlcDAG) were extracted by chloro-

form/methanol (2 : 1, v/v), and typical fluorescence spec-

tra of mono-pyrenyl and bis-pyrenyl glucolipids visualized

(Fig. 2B,C).

Incorporation of PD and synthesis of pyrenyl

glucolipids

in vivo

No growth of A. laidlawii could be observed with only 16:0

or PD, separately or together (Table 1). The presence of

18:1c fatty acid was very important for both the growth of

A. laidlawii and the incorporation of PD into pyrenyl lipids.

However, the yield of pyrenyl lipids was quite low (less than

10% on the basis of added fatty acids) compared to the

nonpyrenyl lipids (30%)40%), but incorporation into

MGlcDAG and DGlcDAG was fairly similar.

The yield of nonpyrenyl MGlcDAG from A. laidlawii

strain A-EF22 was much lower than that of nonpyrenyl

DGlcDAG when PD in growth media; revealed by

quantitation of nonpyrenyl lipids from excised gel spots

(data not shown).

In the same membrane 18:1c fatty acid preferred to

incorporate PD acid to produce mono-pyrenyl glucolipid

rather than nonpyrenyl glucolipid, whereas 16:0 dominated

in the latter (data not shown). The Ie/Im ratio from the

fluorescence spectra increased for the extracted lipid mixture

from cells when increasing the PD ratio in the medium,

showing that more bis-pyrenyl lipids were synthesized at

higher PD acid to fatty acid ratios (data not shown). The

yield of synthesized pyrenyl glucolipids was determined

from standard fluorescence intensity curves, obtained from

synthetic PyrDAG and bis-PyrPG.

Enzymes and assays

Mixed lipid micelles were made by swelling dry lipid to a

final concentration of 10 m

(1 m

substrate) in a buffer of

110 m

Tris pH 8, 22 m

Chaps, 22 m

.Purified

MGlcDAG synthase (50 lL) or DGlcDAG synthase was

incubated with 40 lL lipid micelles at 4 Cfor30min.The

enzyme reaction was started by adding 10 lLof10m

(0.5 CiÆmol

)UDP-[

C]glucose. The reactions were ter-

minated by the addition of 375 lL methanol/chloroform

(2 : 1, v/v). Synthesized MGlcDAG or DGlcDAG was

extracted according to a modified Bligh and Dyer method

[38] and separated from other lipids by TLC. The

C-

labeled glucolipid products were quantified using electronic

autoradiography (Packard Instant Imager). Homogeneous

MGlcDAG synthase for liposome binding was purified

from detergent-solubilized A. laidlawii cells by three column

chromatography methods, including ion exchange, gel

filtration and hydroxyapatite chromatography [39].

Experimental design

Chemometrics is how to design an experimental series in

order to extract the maximum information from the

minimum number of experiments [40]. Chosen factors are

varied simultaneously in a randomized run order to reduce

or eliminate unknown or uncontrolled influence on data.

Table 1. Incorporation of PD and yield of A. laidlawii pyrenyl lipids.

Fatty acids (l

) PD/FA

% Incorporation

of PD into lipids16:0 18:1c PD Initial Harvest

1 120 30 0 – – –

2 90 30 30 0.25 0.024 3.83

3 30 30 90 1.5 0.21 8.13

4 0 60 90 1.5 0.31 6.17

5 0 30 120 4 0.68 7.03

6 0 10 140 14 1.62 4.22

FEBS 2003 Lateral organization of A. laidlawii lipids (Eur. J. Biochem. 270) 1701

The response(s) Yis then fitted to the variables by a

mathematical model, e.g. Y¼m+Xb +e;wereXis the

model terms/variables, bis the coefficient of effect and eis

the residuals.

We have used the

MODDE

3.0 package (Umetri AB,

Umea

˚, Sweden). Here, variables were changed from low to

high and the response was plotted and analyzed in the

computer to give a measure of effects. Variables in this case

are the amounts of different lipid headgroups (as all acyl

chains are 18:1c) and the amount of MGlcDAG synthase.

Responses are excimer formation of pyrene-labeled phos-

pholipid or anisotropy of diphenylhexatriene (DPH).

DGlcDAG, considered the matrix lipid, was set as a filler

and a full factorial design was chosen. In the simple case

of three variables (dimensions, factors), a full factorial

design is a cube in the experimental space, where data points

are in the corners and center of the cube (Fig. 1), resulting

in a linear interaction model. In a couple of cases the

investigation was expanded to a response surface model,

i.e. composite face-centered (CCF) design, where the design-

cube also has data points on the face of the sides, making

quadratic models possible to obtain. For the investigation

of chain ordering for pure lipids a mixture (

-optimal)

design was chosen, where matrix lipid DGlcDAG is not set

as a filler.

Partial least squares (PLS) was used to fit the model.

PLS finds the relationship between a matrix Y(response

variables) and a matrix X(model terms). Measure of model

fit is R

¼1)(RSS/YSS), where RSS is residual sum of

squares and YSS is the response sum of squares. Internal

validation (crossvalidation or prediction ability) is measured

by the Q

value, i.e. Q

¼1)(PRESS/YSS), where PRESS

is the predicted residual sum of squares. Rules of thumb are

that R

should be at least 0.8 and Q

above 0.3 for linear

models and even closer to one for quadratic models. R

and

are the overall parameters for model accuracy, which

encompass analysis of variance (

ANOVA

), lack of fit, normal

distribution of residuals. If these parameters are not

satisfactory, then outliers, wrong metric, inhomogeneous

data, range of the factors, etc., need to be investigated. The

fitted model can then be presented as a response surface

(Fig. 1B), a curve or a table.

Furthermore, an important aspect of experimental design

is that interaction effects can be detected; this would not be

possible if only one variable at a time was changed. Inter-

action means that the response of a variable is dependent on

the level of another variable in a nonadditive fashion.

Preparation of large unilamellar vesicles

For each sample 0.25 lmol of total lipid was mixed to the

desired composition according to the experimental design,

where DOPG was varied 0–40 mol/100 mol, DOPA

0–10 mol/100 mol, MGlcDAG 0–30 mol/100 mol, DOG

0–10 mol/100 mol, and DGlcDAG was used as the (bal-

ance) matrix. The content of fluorescence probe was

constant at 1 mol/100 mol for mono-pyrenyl lipids, and

0.5 mol/100 mol for bis-pyrenyl lipids or DPH. The mixture

was then dried under a nitrogen flow and then under

reduced pressure (vacuum) overnight. The resulting lipid

film was hydrated with intermittent vortexing during 45 min

in filtered and deoxygenated 10 m

Hepes pH 8.0 with

MgCl

, and then extruded with a LiposoFast Basic

extruder (Avestin Inc., Canada) 19 times through two

stacked polycarbonate filters (Millipore; pore diameter

100 nm). This yields large unilamellar vesicles (LUV) with

an average diameter of nearly 100 nm [41]. The quality of

vesicles for all data points was verified with dithionite

quenching of an NBD-probe, showing that all vesicles were

LUVs, as only the outer leaflet is quenched and roughly

50% of the signal remained after quenching (data not

shown).

Fluorescence and absorbance methods

Absorbance measurements were performed with a Beckman

DU 70 spectrophotometer.

Fluorescence measurements with labeled vesicles were

carried out so that 50 lL of prepared liposomes were added

to 1950 lL buffer in an optical 1 ·1 cm fluorescence

cuvette, and fluorescence measured with a Spex Fluoro-

Max-2 fluorometer with magnetic stirrer and temperature

control (28 C). Samples with pyrene probes, were excited at

344 nm and emission spectra collected between 360 and

500 nm. Slits had a bandwidth of 1 nm for excitation and

4 nm for emission (step width 1 nm, integration time 0.5 s).

Four scans were sampled, averaged, and subtracted by a

blank consisting of the buffer, in order to obtain the

fluorescence curve. Vesicles without a probe do not

particularly affect the spectra, as verified in a control design

showing only noise that was virtually the same as the blank;

therefore no such reference was used in any of the runs.

Ie/Im (excimer ratio) was calculated as the ratio between

excimer emission at 480 nm (Ie), when two pyrenes are

in close proximity (3.5 A

˚), and monomer emission at

398 nm (Im). Enzyme (MGlcDAG synthase) was incubated

with 50 lL liposomes (protein : lipid 1 : 700–1 : 70) on ice

for 30 min prior to measurement at room temperature in a

1·0.2 cm quartz fluorescence cuvette using a Perkin-

Elmer LB50 spectrofluorimeter.

DPH anisotropy [42,43] was analyzed using a Spex

Fluorolog 12 fluorometer (Department of Biophysical

Chemistry, Umea

˚University), where bandwidths were

3.6 nm for excitation and 7.2 nm for emission. Sample

Fig. 1. Experimental design. (A) Full factorial design cube with cen-

terpoint. Variables, e.g. lipids, are changed from low to high amounts.

(B) Example of a response surface plot showing response variation

when varying two variables. The purpose of the design is to extract

maximum information from a minimum number of experiments.

1702 P. Storm et al.(Eur. J. Biochem. 270)FEBS 2003

solution was equilibrated for five minutes in the cuvette

holder (no magnetic stirrer) to reach a temperature of 28 C

prior to measurement. Absorption at the excitation wave-

length was less than 0.09, thus a minimal reabsorption.

Anisotropy r¼(I

)/(I

+2I

) for each datapoint was

calculated and averaged in connection to the measurement

by a computer program.

Results

Enzymes recognize pyrene derivatives

For analysis of potential interactions between the various

A. laidlawii membrane lipids we chose fluorescence spectro-

scopy, with pyrene-labeled lipids containing one normal and

one pyrene-labeled chain as proximity probes, as the studied

phenomena may be transient and not possible to isolate,

and too small (<300 nm) to be detected with microscopy.

Pyrene-decanoyl chains locate in the membrane hydro-

phobic core and are virtually nonperturbing at a fraction of

1 mol/100 mol or less, partitioning preferentially in the fluid

phase [44–49]. To investigate chain ordering, i.e. membrane

free volume (V

), a bis-pyrenyl lipid, with a pyrene on both

acyl chains, was used [47,50].

Native A. laidlawii pyrene-labeled glucolipids (not com-

mercially available) were produced in vivo,andtestedas

lipid enzyme substrates in vitro to monitor the impact of the

pyrenyl chain moiety on headgroup organization. Pyrenyl-

decanoic acid (PD) was used for the incorporation of the

pyrene group into the lipids. 16:0 and 18:1c fatty acid were

chosen for their approximately similar chain length to PD.

Different compositions of growth medium fatty acids were

used to optimize the incorporation of PD acid, with or

without 16:0 and 18:1c, into the glucolipids (Table 1). Cell

growth and size of cells were checked by routine light

microscopy. PD or 16:0 could not support growth, alone or

in combination. A. laidlawii cells became much bigger and

less aggregated, and the density of the culture became

lower,whentheratioofPDinfattyacidswasincreased.

One-dimensional thin layer chromatography developed in

chloroform/methanol/water (80 : 25 : 4, v/v/v) was used to

characterize the lipids extracted from the cells (Fig. 2). The

values of different lipids on a TLC plate were compared

according to the standard samples characterized by NMR

[1,51]. Fluorescent spots (under UV light) are marked by

rings in Fig. 2A. Combined with the data from radiolabel

analysis, it is obvious that without a pyrene group in the

hydrocarbon chain of the lipid, there was no fluorescence.

With one PD acyl chain and the other chain 16:0 or 18:1c,

as in mono-pyrenyl lipids, both fluorescence and isotope

signals were detected (data not shown). With two pyrenyl

chains, only fluorescence but no isotope signal could be

detected from the spot of the bis-pyrene lipid on the TLC

plate (Fig. 2A). Note that mono-pyrenyl lipid migrated a

little further than the nonpyrenyl lipid, and bis-pyrenyl

lipid migrated even further, as expected from the larger

hydrocarbon regions of the pyrenyl-containing lipids

(Fig. 2A).

Purified MGlcDAG synthase and partially purified

DGlcDAG synthase were used to study the potential

disturbance of the polar headgroup organization by the

purified pyrenyl-labeled glucolipids in vitro (Fig. 3). The

enzymatic products, pyrenyl-MGlcDAG or pyrenyl-DGlc-

DAG, from the in vitro enzyme reactions, were extracted

from TLC plates. Similar yields were obtained for pyrenyl-

glucolipid and nonpyrenyl-glucolipid products, from both

the MGlcDAG synthase and DGlcDAG synthase reactions

(Fig. 3). Furthermore, the shape of the fluorescence spectra

of the product depends on which type of pyrenyl lipid was

used as substrate; mono- and bis-pyrenyl lipid substrate

produced mono- or bis-pyrenyl glucolipid products,

respectively (data not shown). Thus, these enzymes do not

discriminate between substrates with a pyrene moiety in the

acyl chain. Similar features have been observed for enzymes

Fig. 2. In vivo synthesis of pyrenyl lipids. (A) A. laidlawii

H-

labeled glucolipids and pyrenyl-glucolipids after TLC separation.

Extracted lipids applied on TLC plates were developed in chloroform/

methanol/ammonia (91 : 35 : 10, v/v/v). The growth medium fatty

acid composition (ratio 16:0/18:1c/PD) was from left to right:

120 : 30 : 0; 90 : 30 : 30; 30 : 30 : 90; 0 : 60 : 90; 0 : 30 : 120 and

0 : 10 : 140. Encircled spots represent the fluorescent mono-

pyrenylMGlcDAG (lower) and bis-pyrenylMGlcDAG (upper),

respectively. The fluorescence spectra of purified mono-pyrenyl (B)

and bis-pyrenyl glucolipids (C) produced in vivo.Thesamples(0.1m

lipid) were excited at 344 nm in chloroform/methanol (2 : 1, v/v).

Synthetic mono-pyrenylDAG (B), and bis-pyrenylPG (C), were used

as references.

Fig. 3. Pyrenyl lipids as enzyme substrates in vitro.Synthesis of

DGlcDAG from MGlcDAG and UDP-[

C]glucose by purified

DGlcDAG synthase. The contents of pyrenyl glucolipids were less

than 1% (mol/mol). (s)[

C]DGlcDAG produced from di-18:1c-

MGlcDAG; (m) mono-pyrenyl DGlcDAG produced from mono-

pyrenyl MGlcDAG; and (d) bis-pyrenyl DGlcDAG produced from

bis-pyrenyl MGlcDAG, respectively, by the DGlcDAG synthase.

FEBS 2003 Lateral organization of A. laidlawii lipids (Eur. J. Biochem. 270) 1703

Báo cáo khoa học: Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes

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