Journal of General Microbiology (198l), 124,299-307. Printed in Great Britain
299
The Effect of Alterations in the Fluidity and Phase State of the
Membrane Lipids on the Passive Permeation and Facilitated Diffusion of
Glycerol in Escherichia colt'
By M I C H A E L 0. E Z E t A N D R O N A L D N. McELHANEY*
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
(Received 15 August 1980; revised 12 November 1980)
The passive permeation and facilitated diffusion of glycerol into Escherichia coli K1060, an
unsaturated fatty acid auxotroph, were studied as a function of temperature and membrane
lipid fatty acid composition using a stopped-flow spectrophotometric assay of glycerol
permeation. The relative rates of glycerol passive and mediated entry were both significantly
influenced by the fluidity of the membrane lipids, increasing as the gel to liquid-crystalline
phase transition midpoint temperature of the membrane lipids decreased. The rate of passive
glycerol permeation, but not the rate of glycerol facilitated diffusion, decreased as the
membrane lipids were converted to the gel state. The apparent activation energies for passive
and facilitated diffusion of glycerol, measured in cells whose membrane lipids were in the
liquid-crystalline state, were 15-16 and 10-1 1 kcal mol-', respectively, and neither value was
significantly influenced by the fatty acid composition or fluidity of the membrane lipids. The
mechanistic implications of these observations for the function of the glycerol facilitated
diffusion system of E. coli are discussed.
INTRODUCTION
The relative rates at which glycerol and other polar non-electrolytes passively diffuse into
the simple, cell wall-less prokaryote Acholeplasma laidlawii B have been shown to be
markedly dependent on membrane lipid fatty acid and sterol composition (McElhaney et al.,
1970, 1973; Romijn et al., 1972; de Kruijff et al., 1973). Lipid compositional alterations
which increased membrane lipid fluidity also increased permeation rates proportionally. In
contrast, activation energies of permeation were not dependent on membrane lipid
composition, reflecting instead the dehydration enthalpies of the permeant molecules. Similar
results were previously reported for the permeation of glycerol and other non-electrolytes into
liposomes prepared from a variety of synthetic and natural phospholipids (Demel et al., 1968;
de Gier et al., 1968, 1971; McElhaney et al., 1973). We concluded that glycerol dehydration
and insertion into the hydrophobic core of the lipid bilayer was the rate-limiting and
enthalpy-determining step in the overall permeation process, but that the rate of
dehydration-insertion was increased by entropic factors in more disordered lipid membrane
systems.
When Escherichia coli is grown in the presence of glucose, a catabolite repressor of the
L-a-glycerophosphate regulon, glycerol enters cells primarily by passive diffusion; however,
when E. coli is grown in the presence of glycerol or L-a-glycerophosphate as the sole carbon
and energy source, a facilitated diffusion system for glycerol entry is induced (Sanno et al.,
1968; Richey & Lin, 1972; for review, see Lin, 1976). The objective of this study was to
determine if the glycerol facilitated diffusion system of E. coli would exhibit a dependence on
t Present address: Department of Biochemistry, University of Nigeria, Nsukka, Nigeria.
0022-1287/81/0000-9502$02.00 O 1981 SGM
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M. 0 . E Z E A N D R . N . MC E L HA N E Y
the fluidity and phase state of the membrane lipids similar to that which the passive diffusion
of glycerol into this organism would be expected to exhibit, based on the previous studies of
passive glycerol permeation reviewed above. To this end, the relative rates of glycerol entry
by passive and facilitated diffusion into E. coli K1060, an unsaturated fatty acid auxotroph,
were measured as a function of temperature and of membrane lipid fatty acid composition, in
cells grown in the presence or in the absence of glycerol. The phase state of the membrane
lipids at various temperatures was determined by differential thermal analysis. The effect of
alterations in the fluidity and phase state of the membrane lipids on the entry of glycerol into
E. coli by passive and facilitated diffusion was thus determined.
METHODS
Bacterial strain and growth conditions. Escherichia coli K 1060 is an unsaturated fatty acid auxotroph unable
to synthesize or degrade unsaturated fatty acids; it was originally constructed by Schairer & Overath (1969) and
was the generous gift of David F. Silbert. The organism was grown in medium M63 (Miller, 1972) in the presence
of an unsaturated fatty acid [75 pg ml-l; solubilized with 0.12% (w/v) polyoxyethylene (20) cetyl ether (Brij 58)1,
thiamin (1 pg ml-l), 0.3 % (w/v) Casamino acids (Difco) and either 0.4% (w/v) glycerol or 0.23 % (w/v) xylose
or 0.54 % (w/v) glucose as carbon and energy source. Organisms were first grown overnight in a small volume of
complete growth medium with rapid shaking (approx. 170rev. min-') at 37 OC (39 "C when elaidic
acid-supplemented medium was used) in a New Brunswick Scientific Co. gyrotary shaker bath. The overnight
culture was diluted 100-fold with fresh medium and shaking was continued until the mid-exponential growth phase
was reached; the organisms were then harvested by centrifugation at 4 OC and 6000 g for 10 min in a Sorvall
RC2-B centrifuge.
Determination of relative rates of glycerolpermeation. The relative rates of glycerol permeation in E. coli grown
on glycerol, glucose or xylose were determined by the stopped-flow spectrophotometric technique of
Alemohammad & Knowles (1974) as modified by Eze & McElhaney (1978). This technique involves placing cells
in a hypertonic glycerol solution and following the rate of osmotically driven changes in cell volume with time.
Initially, water moves very rapidly out of the cell due to a high osmotic gradient, the plasma membrane pulls away
from the cell wall, and the cell volume is reduced (plasmolysis). However, as glycerol more slowly enters the cell
moving down its concentration gradient, the osmolarity of the cell cytoplasm increases and water re-enters,
causing the cell volume to increase to its original value. The rate of return to the original volume, which is
proportional to the change of reciprocal absorbance at 550 nm, is a measure of the relative rate of glycerol entry.
In the present experiments a glycerol concentration of 400 mM was used. The preparation of E. coli for the swelling
rate assays, the assays themselves, the calculation of reciprocal relaxation times [ l/z (s-')l, which are equivalent
to the first-order rate constants [ k (s-l)], for glycerol entry, and the equipment utilized in these measurements have
been described in detail previously (Eze & McElhaney, 1978).
Lipid extraction, purijcation and analysis. Escherichia coli inner membrane lipids were extracted by the
method of Bligh & Dyer (1959) as modified by Saito & McElhaney (1977). The extracted lipid was purified by
silicic acid column chromatography and membrane lipid fatty acyl groups were converted to the corresponding
methyl esters and analysed by gas-liquid chromatography as described previously by Saito & McElhaney (1977).
Differential thermal analysis. The thermotropic phase behaviour of the extracted and purified total lipid Fraction
from either plasma membranes or whole cells was monitored on a DuPont 900 Thermal Analyzer as described
previously (McElhaney et al., 1973), except that the membrane lipids were dispersed in ethylene glycol/water (1 : 1,
v/v) to avoid freezing and thawing of the aqueous phase at 0 OC during cooling and heating scans.
R E S U L T S A N D DISCUSSION
Unsaturated fatty acid incorporation by E. coli K1060
Much less exogenous unsaturated fatty acid was incorporated by cells grown on glucose
than by those grown on glycerol as the sole carbon and energy source (data not presented).
Thus, a comparison of the relative rates of glycerol entry into glucose- and glycerol-grown
cells was not feasible, since their fatty acid compositions (and hence probably the rates of
passive glycerol entry) would not be comparable. Therefore, a number of other carbon and
energy sources were examined in order to find one which would provide a fatty acid
composition comparable to that of glycerol-grown cells yet would also act as a catabolite
repressor of the glp regulon. Xylose proved to be a suitable carbon and energy source, since:
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301
Table 1. Fatty acid compositions of the total membrane l@ids of E. coli KI060 grown in the
presence of glycerolplus various exogenous unsaturated fatty acids
Escherichia coli K1060 was grown in M63 medium containing 0.4% (w/v) glycerol and 75 pg ml-I
of the unsaturated fatty acid indicated solubilized in 0.12% (w/v) Brij 58. The cultures were harvested
in the mid-exponential growth phase and the fatty acid compositions of the total membrane lipids were
analysed as described in Methods.
Fatty acids* in total membrane lipids (mol % of total fatty acid present)
,
\
Unsaturated fatty Cyclopropane fatty
Saturated fatty
acids
acids
acids
Total unsaturated
Exogenous unsaturated &&
and cyclopropane
fatty acid
1 6 : l 1 8 : l 18:2 17:0,
19:0,
12:O 14:O 16:O 18:0
fattyacids
a
Elaidic (18 : 1J
Oleic (18 : lc)
Palmitoleic (16 : 1,)
Linoleic (18 :2c,c)
0.1
68.6 57.4 43.7
0.2 1.1
1.3 48.4
-
-
9.5
1.0
3.9
-
1.9
0.5
0.3
0.4
19.7 7.8
12.2 25.7
8.9 37.2
10.9 36.7
1.9
0.4
0.3
0.3
68.7
61.3
53-4
51.8
*Fatty acids are designated by the number of carbon atoms present followed by the number of double
bonds; the subscripts c and t refer to the cis- and trans-configurations, respectively, of the double bonds and
the subscript cp denotes the presence of a cyclopropane ring system.
(i) the membrane lipid fatty acid compositions of cells grown on xylose and glycerol were
comparable; (ii) aerobic glycerol-3-phosphate dehydrogenase activity was absent from the
membranes of both glucose- and xylose-grown cells, but was present in the membranes of
glycerol-grown cells (Eze & McElhaney, 1978), suggesting that the glp regulon was repressed
by growth on xylose; and (iii) the addition of 0.05 pM-CuCl,, a potent inhibitor of the glycerol
facilitated diffusion system in erythrocytes (Stein, 1958), to cells grown on glucose or xylose
did not inhibit glycerol entry into these cells, but did significantly reduce the rate of glycerol
entry into glycerol-grown cells, again indicating that the glycerol transport system was absent
from xylose-grown cells.
The fatty acid compositions of the total membrane lipids from E. coli K1060 grown in the
presence of glycerol, as sole carbon and energy source, plus various single exogenous
unsaturated fatty acids, are shown in Table 1. Of the unsaturated fatty acids tested, elaidic
acid was incorporated to the greatest extent, representing over two-thirds of the total
membrane lipid fatty acids. This trans-monounsaturated octadecenoic acid was not converted
to the corresponding cyclopropane acid by the cells, in contrast to the two cismonounsaturated fatty acids tested. Oleic and palmitoleic acids, plus their corresponding
cyclopropane fatty acid derivatives, were directly incorporated to a lesser extent than was
elaidate, although exogenous oleic and palmitoleic acid (and their cyclopropane derivatives)
still made up well over half of the total membrane lipid fatty acids. Exogenous linoleic acid
was incorporated to a slightly lesser extent than the other exogenous unsaturated fatty acids
and was not converted to the corresponding cyclopropane acid. However, when the small
amounts of the cis-monounsaturated and cyclopropane acids present in cells grown with
linoleic acid were included, the total ‘fluidizing’ fatty acids in the membrane lipids still
exceeded half of the total fatty acyl groups present. Although phospholipids containing
cis-cyclopropane fatty acyl chains to undergo a gel to liquid-crystalline phase transition at
somewhat higher temperatures than do phospholipids containing the corresponding
cis-monounsaturated chains, these cyclopropane fatty acids more closely resemble unsaturated than saturated fatty acids in their thermotropic phase behaviour (Silvius &
McElhaney, 1979). Therefore, the cyclopropane fatty acids are included, along with the
unsaturated fatty acids, as ‘fluidizing’fatty acids in the final column of Table 1.
The spectrum of endogenous saturated fatty acids present in the E. coli K1060 membrane
lipids changed in response to the incorporation of the various exogenous unsaturated fatty
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M. 0. EZE A N D R. N . McELHANEY
acids. Cells grown with elaidic acid exhibited a 14 :0/16 :O ratio of more than 2 - 5 , oleic
acid-grown cells exhibited a 14 :0/16 :O ratio of less than 0.5 and the 14 :0/16 :O ratio in
palmitoleic and linoleic acid-grown cells was about 0.2. A similar variation in the average
chain length of the endogenous saturated fatty acids in response to the incorporation of
exogenous fatty acids of differing physical properties has been reported for A . laidlawii B
(Saito et al., 1977; Silvius et al., 1977). Such a response to exogenous fatty acid
incorporation appears to be an example of metabolic compensation in order to maintain the
fluidity of the membrane lipids as nearly constant as possible. The apparent inverse
relationship between the melting temperature of the exogenous unsaturated fatty acids and
the average chain length of the endogenous saturated fatty acids would provide a mechanism
to antagonize the effect of the incorporation of the exogenous fatty acid on the overall
physical properties of the membrane lipids, since the melting behaviour of the endogenous
saturated fatty acids are markedly chain-length dependent. However, as will be seen below,
this compensatory mechanism was only partially effective, and marked variations in the
fluidity and phase state of the E. coli K1060 membrane lipids were induced by enrichment
with exogenous fatty acids of differing physical properties, as has previously been reported
for other E . coli unsaturated fatty acid auxotrophs (for review, see Cronan & Gelmann,
1975).
Differential thermal analysis of membrane lipids
Due to the substantial amounts ,of water associated with our plasma membrane
preparations from E . coli K 1060 and because the membranes themselves were predominantly
protein, we were unable to obtain good differential thermal analytical curves with our
relatively low-sensitivity instrument. Instead, the total lipids from plasma membranes were
extracted and purified and then dispersed in a minimal amount of ethylene glycol/water (1 : 1,
v/v). The phase transitions observed in isolated E. coli membrane lipids are essentially
identical to the lipid transitions observed in the membranes from which the lipids were derived
(Overath & Trauble, 1973; Sackmann et al., 1973; Trauble & Overath, 1973; Baldassare et
al., 1976), and ethylene glycol does not significantly perturb the phase behaviour of E. coli
membranes or isolated lipids relative to pure water dispersions (Baldassare et al., 1976); thus,
we believe that the differential thermal analysis results reported below accurately reflect the
phase behaviour of the membrane lipids in situ.
Temperature-based differential thermal analysis thermograms of ethylene glycol/water
dispersions of the total membrane lipids from E. coli K1060 grown in the presence of various
unsaturated fatty acids are presented in Fig. 1. The phase transition lower boundary (Ts),
upper boundary ( TL) and midpoint temperature (TIM)
are indicated. All membrane lipid
dispersions exhibited thermotropic gel to liquid-crystalline phase transitions corresponding to
the cooperative hydrocarbon chain melting of the phospholipid molecules arranged in bilayer
form. However, the degree of cooperativity and the position of the phase transition varied
'considerably depending on the nature and degree of incorporation of the exogenous
unsaturated fatty acid. The cooperativity of the transition, as measured by the temperature
range over which the phase transition occurred, decreased in the order 18 : 1, > 18 : 1, >
16: 1, > 18 :2c,c, which was also the order of decreasing phase transition midpoint
temperatures. Membrane lipids enriched in 18 :2c,, exhibited a quite broad endotherm
extending over a range of about 38 "C and showing two peaks, one centred near -7 "C and
another near +17 "C. The existence of two overlapping peaks, which was not noted in the
other lipid dispersions, implies a partial immiscibility of the membrane lipids enriched in
18 :2c,, in the gel state. The order of decreasing cooperativity noted above also corresponds to
the order of decreasing enrichment of the unsaturated fatty acid supplement (and its
cyclopropane derivative, if present) in the membrane lipid (see Table 1). Thus, one reason for
the observed increase in the broadness of the phase transition may relate to the decreasing
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Glycerolpermeation and transport in E. coli
303
18: 1,
TS
18: 1,
Ts
-10 " C
3
TL
+2q "C
Temperature ( O C)
Fig. 1. Temperature-based thermograms of isolated lipids from membranes of E. coli K 1060 grown in
the presence of glycerol plus various unsaturated fatty acids. The temperature differentials between the
lipid dispersions in ethylene glycol/water (1 : 1, v/v) and the inert reference material are plotted as a
function of the temperature of the reference, using a heating rate of 5 OC min-'. Cooling thermograms
were identical except that they were exothermic and shifted 2 to 3 OC toward lower temperatures. The
lower and upper boundaries of the lipid phase transitions are indicated by Ts and TL,respectively, and
the temperature at which the transition is half complete, as measured by the area under the endothermic
peaks, is designated TM.
homogeneity of the membrane lipid fatty acyl groups as one proceeds from the 18 :1, to the
18 :2,,,-enriched preparation. Another factor which may contribute to differences in the
cooperativity relates to the mixing properties of the fatty acyl moieties of the predominant
phospholipid species present. Thus, the 1- 16 :0, 2- 18 :1 and 1- 18 :1t, 2- 18 :1 molecular
species present in 18 :It-enriched lipid dispersions, because of their relatively more similar
physical properties, would be expected to exhibit a more nearly ideal phase mixing behaviour
than would the 1- 16 :0, 2- 14 :0 and 1-16 :0, 2- 18 :2,,c species present in 18 :2 ,,-enriched
membranes. The variation in the phase transition midpoint temperatures with unsaturated
fatty acid supplementation is exactly that expected from the known dependence of the gel to
liquid-crystalline phase transition temperatures of synthetic phospholipid bilayer and
biological membranes on the number and geometrical configurations of double bonds present
and on the relative lengths of the hydrocarbon chains (for reviews, see Cronan & Gelmann,
1975; Melchior & Steim, 1976). Since, in general, membrane lipid fluidity, measured at some
given temperature above the phase transition range, is inversely proportional to the gel to
liquid-crystalline lipid phase transition temperature (for review, see Shinitzky & Barenholz,
1978), one would expect that the fluidity of the various E. coli membranes at, say, 40 "C
would increase in the order 18 : 1, < 18 : 1, < 16 : 1, < 18 :2,,,. However, differential thermal
analysis is a thermodynamic technique for measuring phase state changes and does not
provide a direct measure of membrane lipid 'fluidity'.
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M. 0 . E Z E A N D R . N . M c E L H A N E Y
40
Temperature ( O C)
30
20
10
-1.0
I
I
I
I
(a)
-2.0
0.3
n
d
I
-
W
M
n
-
I
3 0.2
-3.0
I
C
3
-4.0
0.1
-5.0
20
30
40
Temperature ("C)
50
I
I
1
3.2
3.4
3.3
lo3 x l/Temperature (K-')
I
3.5
Fig. 2. (a) Temperature dependence of the rate of passive glycerol permeation into E. coli K1060
grown with xylose plus various unsaturated fatty acids. The rates of passive glycerol entry were
measured as the reciprocal relaxation times (l/z) of cell swelling in hypertonic glycerol. Unsaturated
fatty acids : 0,linoleic acid (1 8 :2,,,); W, palmitoleic acid (1 6 : 1 ,); 0,
oleic acid (1 8 : 1 c ) ; 0,elaidic acid
(18 : I t ) .
(6)Arrhenius plots of the data in (a).
Effect of fatty acid composition and temperature on the rate of glycerol passive diflusion
The relative rates of passive glycerol entry, measured as the reciprocal relaxation times
for cell swelling in hypertonic glycerol, are plotted against temperature in Fig. 2 ( a ) for
E . coli K1060 grown in the presence of xylose and various unsaturated fatty acids. In all
four types of cells there were marked increases in the rates of glycerol permeation with
increasing temperature. In addition, the relative rates of glycerol passive entry were
significantly dependent on the unsaturated fatty acid supplement employed. Glycerol
permeability decreased in the order 18 :2 , , > 16 : 1, > 18 : 1, > 18 :1,. The ratios of the
passive glycerol permeation rates at 40 O C , where the membrane lipids in all cells tested
should be entirely in the liquid-crystalline state, were approximately 2-9 :2.1 : 1.9 : 1.0 for
18 :2,,,-, 16 :lc-, 18 : 1,-and 18 : 1 t-enriched cells, respectively. The relative permeabilities to
glycerol of these four cell types were inversely related to the gel to liquid-crystalline phase
transition midpoint temperatures of their membrane lipids, suggesting that the rate of glycerol
passive diffusion increases with increasing membrane lipid fluidity.
Arrhenius plots of the data reported in Fig. 2(a) revealed regions of apparently linear
relationship between the natural logarithm of the rate of glycerol permeation and the
reciprocal of the absolute temperature (Fig. 2 b), permitting the calculation of apparent
activation energies for the overall passive glycerol permeation process for each cell type over
the temperature ranges studied. For E. coli K 1060 enriched with 18 :2 , , or 16 : l,, the linear
relationship was observed over the entire temperature range of 10 to 45 "C. The apparent
activation energies, calculated from the slope of these lines, were about 15-15.5 kcal mol-'
(1 cal = 4.184 J). However, for 18 :1,- and 18 :1,-enriched cells, a linear relationship was
observed only at temperatures above about 17 "C and 40 "C, respectively; below these
temperatures, the slopes abruptly increased, giving apparent activation energies of 30-3 5 kcal
mol-l, while above these characteristic temperatures similar activation energies (1 5- 16 kcal
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Glycerol permeation and transport in E. coli
305
mol-l) to those noted for 18 :2,,,- or 16 : 1,-enriched cells were seen. Some evidence of a
return to slopes closer to those noted at higher temperatures was exhibited by 18 : 1,- and
18 :1,-enriched cells at temperatures somewhat below the 'break temperature' of 17 "C and
40 O C , respectively. The apparent activation energies for passive glycerol entry into E. coli
K1060 are similar to, but smaller than, those observed for A . laidlawii B (McElhaney et al.,
1973), pig erythrocytes (de Gier et al., 1971) and phospholipid liposomes (de Gier et al.,
1971; McElhaney et al., 1973), where values ranging from 16.8 to 19eOkcal mol-1 and
averaging 18-0- 18 5 kcal mol-I have been reported. Since insignificant levels of the glycerol
facilitator appear to be present in xylose-grown cells, we attribute this modest difference in
activation energy values to undefined species differences (e.g. to the presence of a cell wall in
E. coli) and to differences in the techniques utilized to measure permeation rates.
The Arrhenius break temperatures observed for E . coli K1060 supplemented with 18 : 1, or
18 : 1, may be related to the phase transition behaviour of the membrane lipids. The upper
and lower break temperatures of 18 : 1,enriched cells occurring near 40 and 3 1 "C,
corresponded closely to the upper and lower boundaries of the gel to liquid-crystalline phase
transition temperatures as monitored by differential thermal analysis (Fig. 1). Similarly, the
upper break temperature of 17 OC in 18 : 1,-enriched cells fell between the upper boundary
(+21 "C) and lipid phase transition midpoint temperature (13 "C). Break temperatures in
16 : 1,- and 18 :2,,-enriched cells may not have been observed above 10 "C because of the
lower phase transition temperatures and the reduced cooperativity of the gel to liquidcrystalline lipid phase transitions in the membranes of these cells.
Effect offatty acid composition and temperature on the rate of glycerol facilitated diffusion
The relative rates of mediated glycerol entry, measured as the reciprocal relaxation times
for cell swelling in hypertonic glycerol and corrected for the passive diffusion background,
are plotted against temperature in Fig. 3 (a) for E . coli K1060 grown in the presence of
glycerol and various unsaturated fatty acids. Although all cell types studied exhibited
significant increases in the rate of glycerol facilitated diffusion with temperature, the
temperature dependence of mediated glycerol permeation was considerably smaller than that
observed for passive glycerol entry. The relative rates of mediated glycerol entry were again
found to vary appreciably with the fatty acid composition of the membrane lipids, decreasing
in the order 18 :2,,, > 16 : 1, > 18 : 1, > 18 :l,, as observed for passive glycerol permeation.
At 40 "C, the ratios of the rates of glycerol facilitated diffusion were 2.6 : 1.9 : 1.5 : 1.0 for
18 :2,,,-, 16 : 1,-, 18 : 1,- and 18 : 1,-enriched cells, respectively. These ratios were slightly
lower but similar to those observed for the passive permeation process, indicating that
protein-mediated glycerol transport is also sensitive to the membrane lipid composition. The
inverse relation again observed between the relative rates of glycerol facilitated diffusion and
the gel to liquid-crystalline membrane lipid phase transition temperatures suggests that the
rate of glycerol facilitated diffusion also increases with increasing membrane lipid fluidity, as
previously reported for glucose transport in A . laidlawii B (Read & McElhaney, 1975).
Arrhenius plots of the data reported in Fig. 3 (a) revealed apparently linear relationships
between the natural logarithm of the rate of glycerol facilitated diffusion and the reciprocal of
the absolute temperature over the entire temperature range studied for all fatty acid
compositions tested (Fig. 3 b). In particular, no abrupt breaks in the slopes of the Arrhenius
plots were observed with 18 : 1,- and 18 :ll-enriched cells near the boundaries of the
membrane lipid phase transitions, as previously observed for passive glycerol entry, although
subtle changes in the slopes of these plots might have been masked by an increased scatter of
the experimental points. This result suggests that mediated glycerol permeation is not
markedly sensitive to the phase state of the bulk membrane lipid. The apparent activation
energies for glycerol facilitated diffusion varied little with membrane lipid fatty acid
composition, ranging from 9.5 kcal mol-l for 18 : 1,-enriched cells to 11.3 kcal mol-' for
18 :2,,,-enriched cells; similar behaviour was observed for glucose transport in A . laidlawii B
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-
M . 0 . EZE A N D R. N . McELHANEY
Temperature
-2.2
-2.6
0.12
h
'i
-
(O
C)
-
-
8
-3.0 '
-3.4
0.08
'
0.04
'
-
W
.I-.-.
-3.8
-
-
-4.21
10
20
30
40
Temperature ("C)
50
,
I
,
1
3.2
3.3
3.4
3.5
lo3 x l/Temperature (K-')
Fig. 3. (a) Temperature dependence of the rate of entry of glycerol by facilitated diffusion into E. coli
K1060 grown in the presence of glycerol plus various unsaturated fatty acids. The rates of glycerol
facilitated diffusion were measured as the difference in reciprocal relaxation times (1/z) of cell swelling
between glycerol- and xylose-grown cells. Unsaturated fatty acids : 0, linoleic acid (1 8 :2c,c); W,
palmitoleic acid (16 : lc); 0,oleic acid (18 : lc): 0, elaidic acid (18 : 1J.
(b)Arrhenius plots of the data in (a).
(Read & McElhaney, 1975). These values are significantly lower than the apparent activation
energies of 15-16 kcal mol-' observed for glycerol passive permeation, as expected for a
protein-mediated transport process (Bowyer, 1957). The apparent lack of an effect of the lipid
phase transition on the function of the glycerol facilitator may indicate that this protein
functions as a membrane channel, as has recently been suggested by Heller et al. (1980).
However, the apparent influence of membrane lipid fluidity on the rates of mediated glycerol
permeation would be difficult to explain if this were the case, unless the fatty acid composition
influences the number of functional carriers in the E. coli membrane. Alternatively, the
boundary lipid immediately adjacent to the glycerol carrier protein may remain in a fluid or
semi-fluid state at temperatures where the bulk membrane lipid is solid. The ability of intrinsic
membrane proteins to disorder gel state lipid (for review, see Chapman et al., 1979) and to
retain some function below the phase transition range of the bulk membrane lipid (Silvius &
McElhaney, 1980) has recently been documented.
We thank Mr Steve Cook and Mrs Nannette Mak for excellent technical assistance and Dr Joel Weiner for
valuable discussions and advice. This research was supported by grant MT 4261 from the Medical Research
Council of Canada.
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Glycerolpermeation and transport in E. coli
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