Vol. 64, No. 2, 1975
BIOCHEMICAL
THERMAL
EVENTS
MEMBRANE R.A.
Long,
G. D.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ASSOCIATED
TRANSPORT
Sprott,
WITH
ACTIVE
IN ESCHERICHIA
J. L. Labelle,
W. G.
COLI’:’
Martin
and H. Schneider
Division of Biological Sciences National Research Council of Canada Ottawa, Ontario, Canada KlA OR6 Received
March
25,197s ABSTRACT
Active transport of non-metabolizable compounds by Escherichia coli resulted in thermogenesis. With substrates of the lactose permease (thiomethyl galactoside, lactose) and of the glucose transport system (cu-methylglucoside) the rate of heat production was largest on initial addition, but then decreased. The kinetics of heat production varied with the transport system. For the lactose transport system, more than turnover of the permease was required since heat was not produced in azide treated cells, where facilitated diffusion is known to take place. The lactose permease thermal effects are suggested to reflect operation of the The thermal effects are considered to represent a energy coupling system. useful approach in studying transport energetics and mechanisms.
INTRODUCTION Although the amounts into
involved
transport
measurement have the
not been
processes.
not been
of associated
heat
effects.
bacterial
cells.
made
with
permease
Escherichia
coli.
as NRCC
No.
Q 1975 b-v Academic Press. Im. in arq~ form reserved.
Thus
far,
Insight
be obtained
such
through
measurements
study
investigates
information
about
active
and of the glucose
656
might
energy,
The present
out with
14581.
requires
experimentally.
as mechanism,
in providing
was carried (1,2)
in microorganisms
determined
as well
The study
of reproductiorl
transport
energetics,
the lactose
Copyright All rights
have
of microcalorimetry
utility
+Issued
active
non-metabolizable transport
transport
substrates system
(3) in
of
BIOCHEMICAL
Vol. 64, No. 2,1975
MATERIALS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND
METHODS
Studies of the lactose permease were carried out with --E. coli ML308-225 (i-z-ytat) and ML30 (iSztytas) grown at 37°C in medium 63 (4) IPTG at a concentration of 0. 3 mM was used as inducer plus 0.4% glycerol. with ML30. Studies with the glucose transport system employed ML308-225 grown in medium A (5) supplemented with 0.4% glucose. Cells were harvested in the logarithmic phase of growth, washed twice and resuspended at a concentration of 2 mg cell protein/ml in either medium 63 salts, pH 7.0, or 50 mM potassium phosphate-O. 1 mM with the lactose or glucose transport MgS04 > pH 7. 0, for experiments systems, respectively. Chloramphenicol was added to a final concentration Cells were stored on ice and used within 4 hours after of 80 pg/ml. harvesting.
Lowry
Cell (6).
et.
protein
was
estimated
according
to the procedure
of
Chromatographically pure o-methyl-D-glucopyranoside kindly supplied by Dr. M. B. Perry. Methyl l-thio-P-D-galactopyranoside and lactose were obtained from Sigma Chemical Co.
was
Calorimetric measurements were made with a Tronac Model 550 Isothermal Calorimeter (Tronac Inc. , Orem, Utah, U. S. A. ) employing a 4.0 ml stainless steel reaction vessel. This instrument measures the heat flow required to maintain the contents of the reaction vessel at constant temperature, in this case 25 ‘. The instrument was modified to accommodate a second microburette and also to allow gas to be bubbled through the liquid in the reaction vessel, since air has access to the contents but only via a relatively long and narrow path. Air flow, when employed, was at a rate of 2.1 ml/min. Typically, 3 ml of cells (6 mg protein) were placed in the reaction vessel. After thermal equilibration (approximately 10 min) the baseline was run out for 10 min and then substrate in a volume of 54 ~1 to 120 Pl was added.
RESULTS Lactose
Permease The heat
suspensions bubbled
of E. coli for experiments
Abbreviations:
effects ML308-225
produced
by adding
are shown
a to d, and essentially
TM’;
in Figure
and lactose 1.
Air was
to not
the same results
were
IPTG = isopropyl 1-thio-P-D-galactopyranoside, thiomethyl galactoside, a-MG = cr-methylglucoside, NEM = N-ethylmaleimide.
TMG
657
=
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TMG
TMG
J-A-TLG
lactose
& TMG
TMG
lactose
4-TMG
Figure 1. Thermal profiles associated with the lactose transport system. Sample volume was 3 ml. Additions indicated by arrows were 120 ~1 of 0. 24 M TMG or 54 ~1 of 0. 58 M lactose. Experiments (a), (c), and (d) employed ML308-225, (b) suspending buffer, (e) induced ML30, (f) uninduced M!,30.
obtained if this was done. linear, indicating
Prior to addition of substrate the baselines were
that the rate of endogenous heat production was constant
Addition of 120 ~1 of TMG solution (final concentration
9. 1 mM) initiated
thermogenesis, the rate of which decayed over a period of 15-20 min (Figure 1a).
The heat evolved in the first 20 min in excess of the endogenous
value was = 50 meal.
Further addition of TMG produced a much smaller heat,
z 2 meal, which was comparable to the heat of dilution (Figure lb). of lactose (54 ~1, final concentration
Addition
10. 3 mM) produced a thermal profile
resembling that obtained with TMG (Figure lc).
Prior addition of TMG
prevented heat evolution on adding lactose and vice versa (Figures lc and Id). ~-
658
Vol. 64, No. 2, 1975
BIOCHEMICAL
Addition imately was
the heat
similar
tutive
of dilution.
to that
(Figures
with
facilitated
untreated
cells
ML308-225,
azide
thermic addition,
2.
(2). When
the heat
with
cells
of ML30
induced
produced
cells,
where
the lactose
active
accumulation
approx-
the response
permease
is consti-
The Km for influx TMG
evolved
was
added
of P-galactosides, of poisoned
to cells
was comparable
and
in the presence
to that
for dilution.
System The thermal
Figure
inhibits
diffusion
azide
Transport
concentration
However,
is the same.
of 30 mM sodium Glucose --
to uninduced
RESEARCH COMMUNICATIONS
le and If). Sodium
but allows
of TMG
AND BIOPHYSICAL
5.0 Air was
processes in about
profile
mM) to glucose
obtained grown
bubbled
at the rate
seemed
to occur;
20 seconds;
a
by adding E. coli
CU-MG (80 ~1, final
ML308-225
of 2. 1 ml/min. one peaking
the other
after
Two different
shortly about
is shown
after
3 min.
in exo-
substrate The heat
h
a-MG
a-MG
profiles following addition of PMG to glucose grown Figure 2. Thermal ML308-225. A volume of 80 r~.l of 0.20 M cr-MG was added where indicated by arrows to 3 ml of: (a) cells, (b) suspending buffer, (c) cells plus 30 mM sodium azide.
659
Vol. 64, No. 2, 1975
liberated
for the first
approximately effect
BIOCHEMICAL
15 min in excess
9 0 meal.
comparable
Subsequent
to the heat
(7) and N-ethyl
maleimide
of CPMG
suspensions
gave
to cell
thermal
shown)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
profiles
of that produced addition
of dilution
(8) inhibit
comparable
was
of more LY-MG produced
(= 2 meal, glucose
containing
endogenously
Figure
transport
2b).
Iodoacetate
in E. coli.
4 mM iodoacetate
to the dilution
an
heat
Addition
or 0. 2 mM NEM
of (Y-MG
(data
not
on (r-MG
transport
. Sodium
The thermal in Figure
profile 2c.
quently
heat
before
addition
azide
has no inhibitory
obtained
effect
in the presence
A rapid
exothermic
production
declined
reaction
of 30 mM sodium was
below
observed.
the endogenous
azide
is shown
However, level
(9).
subse-
observed
of (Y-MG.
DISCUSSION The data suggest that microcalorimetric well
be useful
view
depends
in elucidating on the sensitivity One of the more
parameters. with
the nature
of the transport
phosphotransferase processes, effects
transport
system
reflect
are a source
which
effects effort
objective were was
of this specifically
directed
and energetics.
measurements
is the variation
system.
The uptake
p-galactoside of these
occurring
systems
during
of the heat generating
initial
study
associated
at identifying
with
660
of (Y-MG
via the
involves
differ.
process
profile
occurs
other
Thus,
process,
of the events
a transport
the thermogenic
of the thermal
the uptake
was to demonstrate
This
to several
uptake
may aid in elucidation
The nature main
important
profiles
the chemistry of data
of the heat
(3) while
and the thermal
mechanisms
measurements may
the heat and hence
involved.
is not clear. that
process, processes.
A
the thermal and little The dependence
BIOCHEMICAL
Vol. 64, No. 2, 1975
of thermogenesis induction
on a functional
and inhibition
provided
insight
The effects
of the permease operation
reflect
the energy
tion
study
substrate
on TMG
flow
is first
However,
induced
coupling
associated
regard
added,
heats
was
demonstrated
these
experiments
that
the mass
permease.
is the larger
rate
the rate
also
in one instance.
that
That
is,
flow
resulting
A point
by the
more than
suggested
system.
with
or when
process
indicate
It is tentatively
of the lactose in this
system
of the thermogenic
of the energy
of substrates
detailed
the nature
is required.
reflect
transport
experiments.
into
of azide
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
which
the heats
the observed
heats
in accumula-
bears
of thermogenesis
of accumulation
turnover
further when
would
be expected
to be greatest. The possibility the energy interest
coupling since
transport place
which
per mole during
system
it may lead
the processes
that the thermal associated to its
occur.
operation
permease
is of
and to the detailed
nature
evaluation
may provide
process
reflect
the lactose
identification
In addition,
of substrate
the transport
with
effects
insight
of the enthalpy into
the events
of
of
of taking
itself.
ACKNOWLEDGEMENTS We are indebted assistance are also
in showing indebted
to Dr.
that
to Dr.
the quantities
G. C. Kresheck
G. C.
Benson
of interest for advice
and Dr. were
J. L. Fortier
measurable.
for We
and stimulation.
REFERENCES 1. Rickenberg, H. V., Cohen, G. N., Buttin, G., and Monod, J. (1956) Ann. Inst. Pasteur 91, 829-857. 2. Winkler, H. H., and Wilson, T. H. (1966) J. Biol. Chem. 241, 2200-2211. 3. Roseman, S. (1969) in Membrane Proteins, New York Heart Assoc. Symp., Little Brown and Co., Boston, 138-180. 4. Herzenberg, L.A. (1959) Biochim. Biophys. Acta 31, 525-538.
661
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Davis, B. D., and Mingioli, E. S. (1950) J. Bacterial. 60, 17-28, 6. Lowry, 0. H. , Rosebrough, N. J. , Farr, A, L. , and Randall, R. J. (195 1) J. Biol. Chem. --193, 265-275. 7. Klein, W. L., and Boyer, P. D. (1972) J. Biol. Chem. 247, 7257-7265. 8. Haguenauer-Tsapis, R., and Kepes, A. (1973) Biochem. Biophys. Res. Commun. ki4_, 1335-1341. 9. Cohen, G. N., and Monad, J. (1957) Bacterial. Rev. 21, 169-194.
662
BIOCHEMICAL
THERMAL
EVENTS
MEMBRANE R.A.
Long,
G. D.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ASSOCIATED
TRANSPORT
Sprott,
WITH
ACTIVE
IN ESCHERICHIA
J. L. Labelle,
W. G.
COLI’:’
Martin
and H. Schneider
Division of Biological Sciences National Research Council of Canada Ottawa, Ontario, Canada KlA OR6 Received
March
25,197s ABSTRACT
Active transport of non-metabolizable compounds by Escherichia coli resulted in thermogenesis. With substrates of the lactose permease (thiomethyl galactoside, lactose) and of the glucose transport system (cu-methylglucoside) the rate of heat production was largest on initial addition, but then decreased. The kinetics of heat production varied with the transport system. For the lactose transport system, more than turnover of the permease was required since heat was not produced in azide treated cells, where facilitated diffusion is known to take place. The lactose permease thermal effects are suggested to reflect operation of the The thermal effects are considered to represent a energy coupling system. useful approach in studying transport energetics and mechanisms.
INTRODUCTION Although the amounts into
involved
transport
measurement have the
not been
processes.
not been
of associated
heat
effects.
bacterial
cells.
made
with
permease
Escherichia
coli.
as NRCC
No.
Q 1975 b-v Academic Press. Im. in arq~ form reserved.
Thus
far,
Insight
be obtained
such
through
measurements
study
investigates
information
about
active
and of the glucose
656
might
energy,
The present
out with
14581.
requires
experimentally.
as mechanism,
in providing
was carried (1,2)
in microorganisms
determined
as well
The study
of reproductiorl
transport
energetics,
the lactose
Copyright All rights
have
of microcalorimetry
utility
+Issued
active
non-metabolizable transport
transport
substrates system
(3) in
of
BIOCHEMICAL
Vol. 64, No. 2,1975
MATERIALS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND
METHODS
Studies of the lactose permease were carried out with --E. coli ML308-225 (i-z-ytat) and ML30 (iSztytas) grown at 37°C in medium 63 (4) IPTG at a concentration of 0. 3 mM was used as inducer plus 0.4% glycerol. with ML30. Studies with the glucose transport system employed ML308-225 grown in medium A (5) supplemented with 0.4% glucose. Cells were harvested in the logarithmic phase of growth, washed twice and resuspended at a concentration of 2 mg cell protein/ml in either medium 63 salts, pH 7.0, or 50 mM potassium phosphate-O. 1 mM with the lactose or glucose transport MgS04 > pH 7. 0, for experiments systems, respectively. Chloramphenicol was added to a final concentration Cells were stored on ice and used within 4 hours after of 80 pg/ml. harvesting.
Lowry
Cell (6).
et.
protein
was
estimated
according
to the procedure
of
Chromatographically pure o-methyl-D-glucopyranoside kindly supplied by Dr. M. B. Perry. Methyl l-thio-P-D-galactopyranoside and lactose were obtained from Sigma Chemical Co.
was
Calorimetric measurements were made with a Tronac Model 550 Isothermal Calorimeter (Tronac Inc. , Orem, Utah, U. S. A. ) employing a 4.0 ml stainless steel reaction vessel. This instrument measures the heat flow required to maintain the contents of the reaction vessel at constant temperature, in this case 25 ‘. The instrument was modified to accommodate a second microburette and also to allow gas to be bubbled through the liquid in the reaction vessel, since air has access to the contents but only via a relatively long and narrow path. Air flow, when employed, was at a rate of 2.1 ml/min. Typically, 3 ml of cells (6 mg protein) were placed in the reaction vessel. After thermal equilibration (approximately 10 min) the baseline was run out for 10 min and then substrate in a volume of 54 ~1 to 120 Pl was added.
RESULTS Lactose
Permease The heat
suspensions bubbled
of E. coli for experiments
Abbreviations:
effects ML308-225
produced
by adding
are shown
a to d, and essentially
TM’;
in Figure
and lactose 1.
Air was
to not
the same results
were
IPTG = isopropyl 1-thio-P-D-galactopyranoside, thiomethyl galactoside, a-MG = cr-methylglucoside, NEM = N-ethylmaleimide.
TMG
657
=
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TMG
TMG
J-A-TLG
lactose
& TMG
TMG
lactose
4-TMG
Figure 1. Thermal profiles associated with the lactose transport system. Sample volume was 3 ml. Additions indicated by arrows were 120 ~1 of 0. 24 M TMG or 54 ~1 of 0. 58 M lactose. Experiments (a), (c), and (d) employed ML308-225, (b) suspending buffer, (e) induced ML30, (f) uninduced M!,30.
obtained if this was done. linear, indicating
Prior to addition of substrate the baselines were
that the rate of endogenous heat production was constant
Addition of 120 ~1 of TMG solution (final concentration
9. 1 mM) initiated
thermogenesis, the rate of which decayed over a period of 15-20 min (Figure 1a).
The heat evolved in the first 20 min in excess of the endogenous
value was = 50 meal.
Further addition of TMG produced a much smaller heat,
z 2 meal, which was comparable to the heat of dilution (Figure lb). of lactose (54 ~1, final concentration
Addition
10. 3 mM) produced a thermal profile
resembling that obtained with TMG (Figure lc).
Prior addition of TMG
prevented heat evolution on adding lactose and vice versa (Figures lc and Id). ~-
658
Vol. 64, No. 2, 1975
BIOCHEMICAL
Addition imately was
the heat
similar
tutive
of dilution.
to that
(Figures
with
facilitated
untreated
cells
ML308-225,
azide
thermic addition,
2.
(2). When
the heat
with
cells
of ML30
induced
produced
cells,
where
the lactose
active
accumulation
approx-
the response
permease
is consti-
The Km for influx TMG
evolved
was
added
of P-galactosides, of poisoned
to cells
was comparable
and
in the presence
to that
for dilution.
System The thermal
Figure
inhibits
diffusion
azide
Transport
concentration
However,
is the same.
of 30 mM sodium Glucose --
to uninduced
RESEARCH COMMUNICATIONS
le and If). Sodium
but allows
of TMG
AND BIOPHYSICAL
5.0 Air was
processes in about
profile
mM) to glucose
obtained grown
bubbled
at the rate
seemed
to occur;
20 seconds;
a
by adding E. coli
CU-MG (80 ~1, final
ML308-225
of 2. 1 ml/min. one peaking
the other
after
Two different
shortly about
is shown
after
3 min.
in exo-
substrate The heat
h
a-MG
a-MG
profiles following addition of PMG to glucose grown Figure 2. Thermal ML308-225. A volume of 80 r~.l of 0.20 M cr-MG was added where indicated by arrows to 3 ml of: (a) cells, (b) suspending buffer, (c) cells plus 30 mM sodium azide.
659
Vol. 64, No. 2, 1975
liberated
for the first
approximately effect
BIOCHEMICAL
15 min in excess
9 0 meal.
comparable
Subsequent
to the heat
(7) and N-ethyl
maleimide
of CPMG
suspensions
gave
to cell
thermal
shown)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
profiles
of that produced addition
of dilution
(8) inhibit
comparable
was
of more LY-MG produced
(= 2 meal, glucose
containing
endogenously
Figure
transport
2b).
Iodoacetate
in E. coli.
4 mM iodoacetate
to the dilution
an
heat
Addition
or 0. 2 mM NEM
of (Y-MG
(data
not
on (r-MG
transport
. Sodium
The thermal in Figure
profile 2c.
quently
heat
before
addition
azide
has no inhibitory
obtained
effect
in the presence
A rapid
exothermic
production
declined
reaction
of 30 mM sodium was
below
observed.
the endogenous
azide
is shown
However, level
(9).
subse-
observed
of (Y-MG.
DISCUSSION The data suggest that microcalorimetric well
be useful
view
depends
in elucidating on the sensitivity One of the more
parameters. with
the nature
of the transport
phosphotransferase processes, effects
transport
system
reflect
are a source
which
effects effort
objective were was
of this specifically
directed
and energetics.
measurements
is the variation
system.
The uptake
p-galactoside of these
occurring
systems
during
of the heat generating
initial
study
associated
at identifying
with
660
of (Y-MG
via the
involves
differ.
process
profile
occurs
other
Thus,
process,
of the events
a transport
the thermogenic
of the thermal
the uptake
was to demonstrate
This
to several
uptake
may aid in elucidation
The nature main
important
profiles
the chemistry of data
of the heat
(3) while
and the thermal
mechanisms
measurements may
the heat and hence
involved.
is not clear. that
process, processes.
A
the thermal and little The dependence
BIOCHEMICAL
Vol. 64, No. 2, 1975
of thermogenesis induction
on a functional
and inhibition
provided
insight
The effects
of the permease operation
reflect
the energy
tion
study
substrate
on TMG
flow
is first
However,
induced
coupling
associated
regard
added,
heats
was
demonstrated
these
experiments
that
the mass
permease.
is the larger
rate
the rate
also
in one instance.
that
That
is,
flow
resulting
A point
by the
more than
suggested
system.
with
or when
process
indicate
It is tentatively
of the lactose in this
system
of the thermogenic
of the energy
of substrates
detailed
the nature
is required.
reflect
transport
experiments.
into
of azide
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
which
the heats
the observed
heats
in accumula-
bears
of thermogenesis
of accumulation
turnover
further when
would
be expected
to be greatest. The possibility the energy interest
coupling since
transport place
which
per mole during
system
it may lead
the processes
that the thermal associated to its
occur.
operation
permease
is of
and to the detailed
nature
evaluation
may provide
process
reflect
the lactose
identification
In addition,
of substrate
the transport
with
effects
insight
of the enthalpy into
the events
of
of
of taking
itself.
ACKNOWLEDGEMENTS We are indebted assistance are also
in showing indebted
to Dr.
that
to Dr.
the quantities
G. C. Kresheck
G. C.
Benson
of interest for advice
and Dr. were
J. L. Fortier
measurable.
for We
and stimulation.
REFERENCES 1. Rickenberg, H. V., Cohen, G. N., Buttin, G., and Monod, J. (1956) Ann. Inst. Pasteur 91, 829-857. 2. Winkler, H. H., and Wilson, T. H. (1966) J. Biol. Chem. 241, 2200-2211. 3. Roseman, S. (1969) in Membrane Proteins, New York Heart Assoc. Symp., Little Brown and Co., Boston, 138-180. 4. Herzenberg, L.A. (1959) Biochim. Biophys. Acta 31, 525-538.
661
Vol. 64, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5. Davis, B. D., and Mingioli, E. S. (1950) J. Bacterial. 60, 17-28, 6. Lowry, 0. H. , Rosebrough, N. J. , Farr, A, L. , and Randall, R. J. (195 1) J. Biol. Chem. --193, 265-275. 7. Klein, W. L., and Boyer, P. D. (1972) J. Biol. Chem. 247, 7257-7265. 8. Haguenauer-Tsapis, R., and Kepes, A. (1973) Biochem. Biophys. Res. Commun. ki4_, 1335-1341. 9. Cohen, G. N., and Monad, J. (1957) Bacterial. Rev. 21, 169-194.
662
