Vol. 67, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESOLUTION AND RECONSTITUTION OF MITOCHONDRIAL NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE
J. RydstrEm', Section
Received
N. Kanner
and E. Racker
of Biochemistry, Molecular and Cell Cornell University, N.Y., USA
September
Biology,
2,1975
Summary: Nicotinamide nucleotide transhydrogenase from bovine heart mitochondria was solubilized with cholate and partially purified by ammoniumsulphate fractionation and density gradient centrifugation. Compared to submitochondrial particles this preparation contained less than 10% of oligomycin-sensitive ATPase and cytochromes. When reconstituted with purified mitochondrial phosphatidylcholine, the enzyme catalyzed a reduction of NAD+ by NADPH that was stimulated by uncouplers and which showed a concomitent uncoupler-sensitive uptake of the lipophilic anion tetraphenylboron, indicating the generation of a membrane potential. It is concluded that transhydrogenase can energize the vesicles directly without the intervention of ATPase or cytochromes.
INTRODUCTION Nicotinamide catalyzes
the
nucleotide reversible
by NADPH energizes the membrane.
The presence
(cf. -
ref.
Several
a review)
In the present energy-linked purified
for
communication
transhydrogenase transhvdrozenase
Abbreviations:
with
and the reduction
and extent
energy
source,
of NAD+
either
similar are
of this
ATP or
and inhibits to those
currently
proposed
being
considered
interaction.
the problem
of the coupling
was approached
by reconstitution
purified
mitochondria,
The reduction
of NADP+ reduction
phosphorylation, the mechanism
heart
of NADP+ by NADH deenergizes
mechanisms,
FCCP,carbonyl cyanide sodium tetraphenylboron;
1 On leave from the Department Stockholm,Sweden.
bovine
of NAD+ by NADPH.
hypothetical
and photosynthetic 1 for
from
of an additional
the rate
NAD+ reduction. oxidative
reduction
the membrane
respiration,stimulates
for
transhydrogenase
mitochondrial
mechanism
of the
of a partially
phospholipids,
p-trifluoromethoxyphenylhydrzone; TPB-, 1799, a 2:l adduct of hexafluoroacgggpgngnd
of Biochemistry,
Arrhenius
Laboratory,
S-10405
Vol. 67, No. 2, 1975
without
the
BIOCHEMICAL
addition
of an auxilliary
or respiratory
chain.
uptake
of TPB-
as a measure
extent
of stimulation
and ionophores.
was monitored
of the membrane
of the rate
of reduction
observations
indicate
in the reconstituted
purified
oligomycin-sensitive
ATPase
transhydrogenase nor
RESEARCH COMMUNICATIONS
energy-generating
Reconstitution
These
AND BIOPHYSICAL
potential
system,
ATPase
by the uncoupler-sensitive generated,
of NAD+.by that
e.g.
NADPH by uncouplers
a membrane
system which
and by the
potential involves
is
formed
neither
cytochrome.
MATERIALS
AND METHODS
Submitochondrial particles were prepared in the presence of sodium pyrophosphate (SMP)(2),or ofMg2+, Mn2+ and ATP (ETPH) (3), and stored at -70'. Protein was determined by the biuret method (4) or by the Lowry method (5). Transhydrogenase was solubilized by incubating SMP for 30 min. at a protein concentration of 20 mg/ml in a medium containing 2% sodium cholate, 10% saturated ammonium sulphate (pH 8.0), 20 mM sodium tricine (pH 8.0), 3 mM EDTA and 2 mM dithiothreitol. After centrifugation for 40 minutes at 100,000 x g (average) the supernatant was decanted and ammonium sulphate was added (dropwise) to obtain 30% saturation. After 20 minutes at 0°C the centrifugation step was repeated and the supematant was decanted. The saturation of armnonium sulphate was then raised to 38% and the mixture was incubated, centrifuged and decanted as in the previous step. The ammonium sulphate concentration was finally raised to 43% saturation and the precipitate was collected. About 90% of the solubilized transhydrogenase was recovered in this pellet (38-431) which was suspended in a medium containing 20 mM sodium tricine (pH 8.0), 3 mM FDTA and 2 mM dithiothreitol at a protein concentration of 20 mg/ml. The temperature during the purification procedure was kept at O-4”. The preparation was stored in batches of 0.5 ml at -70". Sucrose density gradient centrifugation of 38-43P was carried out in a Beckman SW41 rotor (or a Beckman SW50.2 rotor) with a linear gradient of 20 to 50% sucrose. In addition, the medium contained 0.1% purified mitochondrial phosphatidylcholine, 1.5% sodium cholate, 50 mM tris acetate (pH7.4), 1.5 mM EDTA and 2 mM dithiothreitol. One ml 38-43P was layered on top of the chilled gradient and centrifuged for 12 hours at maximal speed (with the SW 50.2 rotor, 0.5 ml of 38-43P and 6 hours of centrifugation was used). After centrifugation, fractions were collected from the bottom of the tube, and activities were assayed imediately. The most active transhydrogenase fractions were pooled, diluted twice with 50 mM tris acetate (pH 7.4), 1.5 mM EDTA and 2 mM dithiothreitol, and concentrated approximately 10 times in an Amicon ultrafiltration cell (Amicon Corp., Lexington, Mass., USA) equipped with a PM 10 filter. Reduction of NAD+ by NADPH &ith lactate-dehydrogenase plus pyruvatej NADH dehydrogenase, succinate-dehydrogenase and ATPase (in the absence and presence of 1 ug oligomycin) were assayed as described earlier (6). Cytochromes were estimated according to Williams (7). Unless otherwise stated, reconstitution of transhydrogen ase in liposomes was carried out essentially as described by Ragan and Racker (8) with 4 mg protein and 40 pmc,les phospholipid per ml. Uptake of TPB- was monitored according to Grinius et al (9), with a medium that contained 50 mM tris acetate (pH 7.4), 1.5 mM EDTA and 0.5 ~.IM tetraphenylboron. The uptake is expressed as the diffusion potential (mV) formed across the lipid membrane separating the two compartments of the instrument. The amount of FCCP (1 pg) used for uncoupling did not affect the permeability of the lipid membrane to TPB-. Mitochondrial phospholipids were extracted and purified as described by Ragawa Cardiolipin (bovine heart) was purchased from Grant Island (10). --et.al. Biological Company (Grand Island, New York, USA). Other biochemicals were purchased from Sigma Chemical Co. (St. Louis, USA) or from Boehringer Mannheim GmbH (Mannheim, Germany).
83%
Vol. 67, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
833
Vol. 67, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS As indicated saturation
(SIG'),
contained
partially
sucrose
3-fold.
density
and cholate
Further
gradient
Activities
7 times.
most
of the
purification
of ATPase
NADH dehydro-
of cytochrome
a and some
of succinate 38-43P
in the presence
in an overall
resulted
of submitochondrial
The activity
fractionation
centrifugation
38 to 43%
transhydrogenase,
whereas
removed.
between
extract
purified
ATPase,
b and CL + c were
was decreased
obtained
in a 2% cholate
and oligomycin-sensitive
of cytochromes
than
1 the precipitate
of ammonium sulphate
particles genase
in Table
dehydrogenase
precipitate
by
of phosphatidylcholine
of transhydrogenase
and succinate
dehydrogenase
of more
were
lower
than
NADPH
i
u
t FCCP
t oligo
1omv __lmin
Fig.
1
Uptake
of TPB-
reconstituted The sample (cf. -
Methods),
compartment purified
a membrane and the
(A),
tuted
gradient-purified
After
equilibration
0.12
for
by NAUPH was added,
oligomycin
was applied
reference
particles
was filled
to the opening
compartment
with
sample
compartment.
for
reduction
of NAU+
system
The TPB- uptake
0.5 mM NAD' or 0.2 mM NADPH.
834
of a partially
to the
NAU+ or NAUPH.
and 1 ng FCCP.
solution
sample
was added
the assay
of either
the
(B) or 0.06
about
either
connecting
38-43P
(C),
2 minutes,
3 ml of medium
and 0.1 mg submitochondrial
phospholipids,
mg of reconstituted
with
a 2%decane
transhydrogenase
except
(A) and
(B,C)
of the instrument
of mitochondrial
(HTPH)
by the addition
by submitochondrial
transhydrogenase compartment
extract
particles
catalyzed
I
Additions
mg of reconsti-
was initiated were
1 pg
BIOCHEMICAL
Vol. 67, No. 2, 1975
10% as compared cytochromes chondrial
to submitochondrial
a,b and cl
+ c were
particles. decreased
Likewise, to less
than
the amounts
of
10% of those
of submito-
particles.
When reconstituted 38-43
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
P fraction
catalyzed
(Fig.
a reduction
similar
to that submitochondrial
than
with
either
ion
lactate
1B) and the
gradient-purified
preparation
(Fig.
with
not
from
three
TPB- uptake 1).
alter
particles
the uptake
latter
systems.
of TPB-
was abolished
Addition
the reduction
reversed
of NAB+ by NADPH (not
shown,
or
of addition shown).
oligomycin
of In all
was without
the uptake ref.
of the
rotenone
(not
cf.
IA).
because
of either
significantly
of 1 mM NADP+ also
of TPB-,
(Fig.
of the order
by FCCP whereas
1C)
more transient
presumably
or reversal
uptake
(ETPH)
Omission
the
by uptake
was considerably systems,
the medium, the
was paralleled
submitochondrial
two reconstituted of the
did
inhibiting
both
of the
substrates,
(Fig.
phospholipids
particles
dehydrogenase
effect
mitochondrial
of NAB+ by NADPH which
permeability
cases
purified
observed
With
lower
with
of TPB- by
11).
Fig.
2
20 > E I
14
5:o
4:1
I
I
I
3:2
2:3
1:4
’ lo 0:5
PEA/PC
Fig.
2
Effect
on uptake
of different
of TPB-
by reconstituted TPBThe assay
uptake
(0)
phosphatidylethanolamine/phosphosphatidylcholine
and on the role
of reduction
ratios
of NAB+ by NADPH
(0)
catalyzed
transhydrogenase. was assayed
of reduction
essentially
as described
of NAB+ by NADPH was carried
FCCP. 835
in the out in
legend
the presence
of Fig.
1B.
of 1 ug
Vol. 67, No. 2, 1975
shows
the effect
reconstituted the best about
vesicles. system
tidylcholine
lipid tory
of phospholipid
for
10 times
on the
(not
less
either
Maximal
absence
As shown
valinomycin, presence
uncouplers
valinomycin.
3
Effect
catalyzed
TPB- uptake with
valinomycin,
for
by reconstituted
essentially
Cardiolipin,
whereas
was
without
effect
or combinations
with
of this
were a 5-fold
no significant
inhibi-
excess uptake
of
was ob-
of NAD+ by NADPH, catalyzed
was stimulated
except
to various
extent
and valinomycin
plus
of these a slight
when no phospholipids
’
’
2
4
of different
to be
phosphatidylethanolamine/phospha-
of reduction
(ETPH)
Similarly,
3),
in
phospholipids.
effect
12-
Fig.
the
proved
or phosphatidylcholine,
No significant
particles
of TPB- uptake
phosphatidylethanolamine
TPB- uptake,
(Fig.
2 the rate
plus
extent
of TPB- was achieved
protein
transhydrogenase
of KCl.
chondrial
uptake
of added
in Table
reconstituted
pure
of NAD+ by NADPH.
over
in the
Decreasing
phosphatidylethanolamine
shown).
RESEARCH COMMUNICATIONS
phosphatidylcholine
whereas
to increasing
of reduction
on the
mitochondrial
efficient.
phosphatidylcholine served
Pure
lead
AND BIOPHYSICAL
composition
reconstitution,
ratio
rate with
BIOCHEMICAL
agents
were
by uncouplers, nigericin,
in the
was seen with
stimulation
submito-
by uncouplers
added
during
by
the
and
reconsti-
’
6 8 10 PL/protein(pmol/mg)
phospholipid
12
14
(PL)/protein
20
ratios
on uptake
of TPB-
transhydrogenase.
was assayed
essentially
as described
0.1 mg 38-43P.
836
in
the legend
of Fig.
1
contained
the
of uncouplers
mg reconstituted
P
fraction
gradient-purified
Reconstituted
38-43
Reconstituted
particles
Submitochondrial
Preparation
and 0.06
mM 1799,
reduction
60
61
85
80
133
FCCP
2
0.3
72
70
135
109
101
135
+ nig.
respectively.
val.
120
105
145
+ 1799
particles
(ETPH),
Additions
by submitochondrial
and 15 mM KCl.
0.1 mg submitochondrial
val. val. nmoles NADP'/min./mg
Addition
fraction,
pg nigericin,
Methods)
transhydrogenase
of NAD+ by NADPH catalyzed
of NAD+ by NADPH (cf --
and reconstituted
on reduction
P and gradient-purified
131
none
38-43
0.1
for
particles
and ionophores
assay
1 pg FCCP, 0.3 pg valinomycin,
Medium
Effect
Table
0.12
were mg
Vol. 67, No. 2, 1975
tution did
BIOCHEMICAL
procedure, not
transhydrogenase
catalyze
an uptake
of 15 mM inhibited the presence
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was not
of TPB-
(cf. -
Fig.
transhydrogenase
of FCCP (not
shown).
stimulated 3).
about
in the
the activity
transhydrogenase,
presumably
due to a partial
inactivation
Alternative
reconstitution
procedures
are
show that
partially
being
investigated.
found
and
absence
and in
of the reconstituted
was generally
procedure.
that
agents
at a concentration
transhydrogenase
tution
than
KC1 alone
50%, both
However,
lower
by uncoupling
with
the
nonreconstituted
during
the
reconsti-
therefore
presently
DISCUSSION
The data
presented
and reconstituted stimulated TPB-,
indicating
(12).
the
on the
the
not
for
specific
transhydrogenase.
or a subunit
potential linked
of either
by the
not
reconstituted
transhydrogenase
Ragan and Widger
(16)
yet
reported
recently 838
between
the generation
the
reconstitution
10% are
a minor
cytochromes,
linked
before
a more
has been
of a membrane
of transport
by Mitchell
than
reconstituted
transhydrogenase
se as proposed
by
components
by the
or the
system may be indicative per
these
can not be eliminated
shown directly
of
of cytochromes
interaction (13,14)
unlikely
was less
seems that
of the reconstitutiblyactive
Although
to the
the ATPase potential,
amount
potential
effect
in the reconsti-
reconstitution
molecular
by Skulachev
rendered
omitted
and the
it
and energy
by a direct
is
were
of a membrane
of a membrane
purification
accomplished.
particles,
a possible
potential
of
TPB uptake
demonstrated
protein
activity
uptake That
reaction
used for
generation
Still,
to the generation extensive
ATPase
preparation
the
been
transhydrogenase
to submitochondrial
essential
component
has already
when phospholipids
the transhydrogenase
as compared
an uncoupler-
potential.
of membrane
transhydrogenase
of stimulation
Since
generation
in general of the
agents
absence
catalyzes
of a membrane
to the
A stimulation
tution. in
the generation
in membranes
uncoupling
transhydrogenase
purified
of NAD+ by NADPH and an uncoupler-sensitive
to be related
coupling
communication
mitochondrial
reduction
appears
in this
of protons
(15). of ATP-driven
Vol. 67, No. 2, 1975
BIOCHEMICAL
transhydrogenase
using
transhydrogenase.
It
preparation
Complex
activity
of 4, probably
found
Complex
In contrast
demonstrated
in this
the
lipid
transhydrogenase,
to be optimal
reductase)
as the source
1,reconstituted (hydrophobic
to the paper,
a phosphatidylethanolamine
reflecting
RESEARCH COMMUNICATIONS
with protein)
values
authors
of the ATPase
found
in the reconstitution
a
of the Pi-ATP
ratio
system
the same phospholipid
of
with
to phosphatidylcholine
requirement since
these
a crude
reduction
optimal
of
and soybean
oligomycin-sensitive
by ATP.
with
of the
ATPase
an uncoupler-and
phosphatidylcholine
that
that
of oligomycin-sensitive
NADP' by NADH, driven
than
I (NADH-ubiquinone
was shown
phospholipida,catalyzed
maximal
AND BIOPHYSICAL
ratio
exchange
rather has been
(10).
ACKNOWLEDGEMENT This Biology Dr.
work
was carried
Organization
C. Miller
for
out during
fellowship his
advice
the
awarded regarding
tenure
of a European
to J.R.
The authors
Molecular wish
to thank
the TPB- measurements.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Rydstrzm, J., Hoek, J.B. and Ernster, L. (1975) The Enzymes, in press Racker, E. (1962) Proc. Natl. Acad. Sci. USA, 68, 1659-1663 Beyer, R.E. (1967) Methods in Enzymology I.& 186-194 Jacobs, E.E., Jacob, M., Sanadi, D.R. and Bradley, L.B. (1956) J. Biol. Chem. 223, 147-156. Lowry, O.H., Rosebrough,N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Hoek, J.B. and Hundal, T. (1974) Biochem.Biophys.Res.Commun. Rydstrom,J., 6CJ 448-455. Williams, J.N. (1964) Arch. Biochem. Biophys. 107, 537-543. Ragan, C.I. and Racker, E. (1973) J. Biol. Chem. 248, 2563-2569. Grinius,L.L., Jasaitis, A.A., Kadziauskas, Y.P., Liberman, E.A., Skulachev, V.P., Topali,V.P., Tsofina, L.M. and Vladimirova, M.A. (1970) Biochim. Biophys. Acta 216, l-12. Kagawa, Y., Kandrach, A. and,,Racker, E. (1973) J. Biol. Chem. 248, 676-684. Teixeira da Cruz, A., Rydstrom, J. and Ernster, L. (1971) Eur. J. Biochem. 3, 203-211. Skulachev, V.P. (1971) in Current Topics in Bioenergetics, ~01.4, Academic Press, New York and Lon‘;T;;n, p. 127-185. Asami, K., Juntti, K. and Ernster, L. (1970) Biochim. Biophys. Acta 205, 307-311. Ernster, L., Juntti, K. and Asami, K. (1972) Bioenergetics 5, 351-357. Mitchell, P. (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research,Bodmin. Ragan, C.I. and Widger, W.R. (1975) Biochem. Biophys. Res. Commun. 62, 744749.
839
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESOLUTION AND RECONSTITUTION OF MITOCHONDRIAL NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE
J. RydstrEm', Section
Received
N. Kanner
and E. Racker
of Biochemistry, Molecular and Cell Cornell University, N.Y., USA
September
Biology,
2,1975
Summary: Nicotinamide nucleotide transhydrogenase from bovine heart mitochondria was solubilized with cholate and partially purified by ammoniumsulphate fractionation and density gradient centrifugation. Compared to submitochondrial particles this preparation contained less than 10% of oligomycin-sensitive ATPase and cytochromes. When reconstituted with purified mitochondrial phosphatidylcholine, the enzyme catalyzed a reduction of NAD+ by NADPH that was stimulated by uncouplers and which showed a concomitent uncoupler-sensitive uptake of the lipophilic anion tetraphenylboron, indicating the generation of a membrane potential. It is concluded that transhydrogenase can energize the vesicles directly without the intervention of ATPase or cytochromes.
INTRODUCTION Nicotinamide catalyzes
the
nucleotide reversible
by NADPH energizes the membrane.
The presence
(cf. -
ref.
Several
a review)
In the present energy-linked purified
for
communication
transhydrogenase transhvdrozenase
Abbreviations:
with
and the reduction
and extent
energy
source,
of NAD+
either
similar are
of this
ATP or
and inhibits to those
currently
proposed
being
considered
interaction.
the problem
of the coupling
was approached
by reconstitution
purified
mitochondria,
The reduction
of NADP+ reduction
phosphorylation, the mechanism
heart
of NADP+ by NADH deenergizes
mechanisms,
FCCP,carbonyl cyanide sodium tetraphenylboron;
1 On leave from the Department Stockholm,Sweden.
bovine
of NAD+ by NADPH.
hypothetical
and photosynthetic 1 for
from
of an additional
the rate
NAD+ reduction. oxidative
reduction
the membrane
respiration,stimulates
for
transhydrogenase
mitochondrial
mechanism
of the
of a partially
phospholipids,
p-trifluoromethoxyphenylhydrzone; TPB-, 1799, a 2:l adduct of hexafluoroacgggpgngnd
of Biochemistry,
Arrhenius
Laboratory,
S-10405
Vol. 67, No. 2, 1975
without
the
BIOCHEMICAL
addition
of an auxilliary
or respiratory
chain.
uptake
of TPB-
as a measure
extent
of stimulation
and ionophores.
was monitored
of the membrane
of the rate
of reduction
observations
indicate
in the reconstituted
purified
oligomycin-sensitive
ATPase
transhydrogenase nor
RESEARCH COMMUNICATIONS
energy-generating
Reconstitution
These
AND BIOPHYSICAL
potential
system,
ATPase
by the uncoupler-sensitive generated,
of NAD+.by that
e.g.
NADPH by uncouplers
a membrane
system which
and by the
potential involves
is
formed
neither
cytochrome.
MATERIALS
AND METHODS
Submitochondrial particles were prepared in the presence of sodium pyrophosphate (SMP)(2),or ofMg2+, Mn2+ and ATP (ETPH) (3), and stored at -70'. Protein was determined by the biuret method (4) or by the Lowry method (5). Transhydrogenase was solubilized by incubating SMP for 30 min. at a protein concentration of 20 mg/ml in a medium containing 2% sodium cholate, 10% saturated ammonium sulphate (pH 8.0), 20 mM sodium tricine (pH 8.0), 3 mM EDTA and 2 mM dithiothreitol. After centrifugation for 40 minutes at 100,000 x g (average) the supernatant was decanted and ammonium sulphate was added (dropwise) to obtain 30% saturation. After 20 minutes at 0°C the centrifugation step was repeated and the supematant was decanted. The saturation of armnonium sulphate was then raised to 38% and the mixture was incubated, centrifuged and decanted as in the previous step. The ammonium sulphate concentration was finally raised to 43% saturation and the precipitate was collected. About 90% of the solubilized transhydrogenase was recovered in this pellet (38-431) which was suspended in a medium containing 20 mM sodium tricine (pH 8.0), 3 mM FDTA and 2 mM dithiothreitol at a protein concentration of 20 mg/ml. The temperature during the purification procedure was kept at O-4”. The preparation was stored in batches of 0.5 ml at -70". Sucrose density gradient centrifugation of 38-43P was carried out in a Beckman SW41 rotor (or a Beckman SW50.2 rotor) with a linear gradient of 20 to 50% sucrose. In addition, the medium contained 0.1% purified mitochondrial phosphatidylcholine, 1.5% sodium cholate, 50 mM tris acetate (pH7.4), 1.5 mM EDTA and 2 mM dithiothreitol. One ml 38-43P was layered on top of the chilled gradient and centrifuged for 12 hours at maximal speed (with the SW 50.2 rotor, 0.5 ml of 38-43P and 6 hours of centrifugation was used). After centrifugation, fractions were collected from the bottom of the tube, and activities were assayed imediately. The most active transhydrogenase fractions were pooled, diluted twice with 50 mM tris acetate (pH 7.4), 1.5 mM EDTA and 2 mM dithiothreitol, and concentrated approximately 10 times in an Amicon ultrafiltration cell (Amicon Corp., Lexington, Mass., USA) equipped with a PM 10 filter. Reduction of NAD+ by NADPH &ith lactate-dehydrogenase plus pyruvatej NADH dehydrogenase, succinate-dehydrogenase and ATPase (in the absence and presence of 1 ug oligomycin) were assayed as described earlier (6). Cytochromes were estimated according to Williams (7). Unless otherwise stated, reconstitution of transhydrogen ase in liposomes was carried out essentially as described by Ragan and Racker (8) with 4 mg protein and 40 pmc,les phospholipid per ml. Uptake of TPB- was monitored according to Grinius et al (9), with a medium that contained 50 mM tris acetate (pH 7.4), 1.5 mM EDTA and 0.5 ~.IM tetraphenylboron. The uptake is expressed as the diffusion potential (mV) formed across the lipid membrane separating the two compartments of the instrument. The amount of FCCP (1 pg) used for uncoupling did not affect the permeability of the lipid membrane to TPB-. Mitochondrial phospholipids were extracted and purified as described by Ragawa Cardiolipin (bovine heart) was purchased from Grant Island (10). --et.al. Biological Company (Grand Island, New York, USA). Other biochemicals were purchased from Sigma Chemical Co. (St. Louis, USA) or from Boehringer Mannheim GmbH (Mannheim, Germany).
83%
Vol. 67, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
833
Vol. 67, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS As indicated saturation
(SIG'),
contained
partially
sucrose
3-fold.
density
and cholate
Further
gradient
Activities
7 times.
most
of the
purification
of ATPase
NADH dehydro-
of cytochrome
a and some
of succinate 38-43P
in the presence
in an overall
resulted
of submitochondrial
The activity
fractionation
centrifugation
38 to 43%
transhydrogenase,
whereas
removed.
between
extract
purified
ATPase,
b and CL + c were
was decreased
obtained
in a 2% cholate
and oligomycin-sensitive
of cytochromes
than
1 the precipitate
of ammonium sulphate
particles genase
in Table
dehydrogenase
precipitate
by
of phosphatidylcholine
of transhydrogenase
and succinate
dehydrogenase
of more
were
lower
than
NADPH
i
u
t FCCP
t oligo
1omv __lmin
Fig.
1
Uptake
of TPB-
reconstituted The sample (cf. -
Methods),
compartment purified
a membrane and the
(A),
tuted
gradient-purified
After
equilibration
0.12
for
by NAUPH was added,
oligomycin
was applied
reference
particles
was filled
to the opening
compartment
with
sample
compartment.
for
reduction
of NAU+
system
The TPB- uptake
0.5 mM NAD' or 0.2 mM NADPH.
834
of a partially
to the
NAU+ or NAUPH.
and 1 ng FCCP.
solution
sample
was added
the assay
of either
the
(B) or 0.06
about
either
connecting
38-43P
(C),
2 minutes,
3 ml of medium
and 0.1 mg submitochondrial
phospholipids,
mg of reconstituted
with
a 2%decane
transhydrogenase
except
(A) and
(B,C)
of the instrument
of mitochondrial
(HTPH)
by the addition
by submitochondrial
transhydrogenase compartment
extract
particles
catalyzed
I
Additions
mg of reconsti-
was initiated were
1 pg
BIOCHEMICAL
Vol. 67, No. 2, 1975
10% as compared cytochromes chondrial
to submitochondrial
a,b and cl
+ c were
particles. decreased
Likewise, to less
than
the amounts
of
10% of those
of submito-
particles.
When reconstituted 38-43
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
P fraction
catalyzed
(Fig.
a reduction
similar
to that submitochondrial
than
with
either
ion
lactate
1B) and the
gradient-purified
preparation
(Fig.
with
not
from
three
TPB- uptake 1).
alter
particles
the uptake
latter
systems.
of TPB-
was abolished
Addition
the reduction
reversed
of NAB+ by NADPH (not
shown,
or
of addition shown).
oligomycin
of In all
was without
the uptake ref.
of the
rotenone
(not
cf.
IA).
because
of either
significantly
of 1 mM NADP+ also
of TPB-,
(Fig.
of the order
by FCCP whereas
1C)
more transient
presumably
or reversal
uptake
(ETPH)
Omission
the
by uptake
was considerably systems,
the medium, the
was paralleled
submitochondrial
two reconstituted of the
did
inhibiting
both
of the
substrates,
(Fig.
phospholipids
particles
dehydrogenase
effect
mitochondrial
of NAB+ by NADPH which
permeability
cases
purified
observed
With
lower
with
of TPB- by
11).
Fig.
2
20 > E I
14
5:o
4:1
I
I
I
3:2
2:3
1:4
’ lo 0:5
PEA/PC
Fig.
2
Effect
on uptake
of different
of TPB-
by reconstituted TPBThe assay
uptake
(0)
phosphatidylethanolamine/phosphosphatidylcholine
and on the role
of reduction
ratios
of NAB+ by NADPH
(0)
catalyzed
transhydrogenase. was assayed
of reduction
essentially
as described
of NAB+ by NADPH was carried
FCCP. 835
in the out in
legend
the presence
of Fig.
1B.
of 1 ug
Vol. 67, No. 2, 1975
shows
the effect
reconstituted the best about
vesicles. system
tidylcholine
lipid tory
of phospholipid
for
10 times
on the
(not
less
either
Maximal
absence
As shown
valinomycin, presence
uncouplers
valinomycin.
3
Effect
catalyzed
TPB- uptake with
valinomycin,
for
by reconstituted
essentially
Cardiolipin,
whereas
was
without
effect
or combinations
with
of this
were a 5-fold
no significant
inhibi-
excess uptake
of
was ob-
of NAD+ by NADPH, catalyzed
was stimulated
except
to various
extent
and valinomycin
plus
of these a slight
when no phospholipids
’
’
2
4
of different
to be
phosphatidylethanolamine/phospha-
of reduction
(ETPH)
Similarly,
3),
in
phospholipids.
effect
12-
Fig.
the
proved
or phosphatidylcholine,
No significant
particles
of TPB- uptake
phosphatidylethanolamine
TPB- uptake,
(Fig.
2 the rate
plus
extent
of TPB- was achieved
protein
transhydrogenase
of KCl.
chondrial
uptake
of added
in Table
reconstituted
pure
of NAD+ by NADPH.
over
in the
Decreasing
phosphatidylethanolamine
shown).
RESEARCH COMMUNICATIONS
phosphatidylcholine
whereas
to increasing
of reduction
on the
mitochondrial
efficient.
phosphatidylcholine served
Pure
lead
AND BIOPHYSICAL
composition
reconstitution,
ratio
rate with
BIOCHEMICAL
agents
were
by uncouplers, nigericin,
in the
was seen with
stimulation
submito-
by uncouplers
added
during
by
the
and
reconsti-
’
6 8 10 PL/protein(pmol/mg)
phospholipid
12
14
(PL)/protein
20
ratios
on uptake
of TPB-
transhydrogenase.
was assayed
essentially
as described
0.1 mg 38-43P.
836
in
the legend
of Fig.
1
contained
the
of uncouplers
mg reconstituted
P
fraction
gradient-purified
Reconstituted
38-43
Reconstituted
particles
Submitochondrial
Preparation
and 0.06
mM 1799,
reduction
60
61
85
80
133
FCCP
2
0.3
72
70
135
109
101
135
+ nig.
respectively.
val.
120
105
145
+ 1799
particles
(ETPH),
Additions
by submitochondrial
and 15 mM KCl.
0.1 mg submitochondrial
val. val. nmoles NADP'/min./mg
Addition
fraction,
pg nigericin,
Methods)
transhydrogenase
of NAD+ by NADPH catalyzed
of NAD+ by NADPH (cf --
and reconstituted
on reduction
P and gradient-purified
131
none
38-43
0.1
for
particles
and ionophores
assay
1 pg FCCP, 0.3 pg valinomycin,
Medium
Effect
Table
0.12
were mg
Vol. 67, No. 2, 1975
tution did
BIOCHEMICAL
procedure, not
transhydrogenase
catalyze
an uptake
of 15 mM inhibited the presence
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was not
of TPB-
(cf. -
Fig.
transhydrogenase
of FCCP (not
shown).
stimulated 3).
about
in the
the activity
transhydrogenase,
presumably
due to a partial
inactivation
Alternative
reconstitution
procedures
are
show that
partially
being
investigated.
found
and
absence
and in
of the reconstituted
was generally
procedure.
that
agents
at a concentration
transhydrogenase
tution
than
KC1 alone
50%, both
However,
lower
by uncoupling
with
the
nonreconstituted
during
the
reconsti-
therefore
presently
DISCUSSION
The data
presented
and reconstituted stimulated TPB-,
indicating
(12).
the
on the
the
not
for
specific
transhydrogenase.
or a subunit
potential linked
of either
by the
not
reconstituted
transhydrogenase
Ragan and Widger
(16)
yet
reported
recently 838
between
the generation
the
reconstitution
10% are
a minor
cytochromes,
linked
before
a more
has been
of a membrane
of transport
by Mitchell
than
reconstituted
transhydrogenase
se as proposed
by
components
by the
or the
system may be indicative per
these
can not be eliminated
shown directly
of
of cytochromes
interaction (13,14)
unlikely
was less
seems that
of the reconstitutiblyactive
Although
to the
the ATPase potential,
amount
potential
effect
in the reconsti-
reconstitution
molecular
by Skulachev
rendered
omitted
and the
it
and energy
by a direct
is
were
of a membrane
of a membrane
purification
accomplished.
particles,
a possible
potential
of
TPB uptake
demonstrated
protein
activity
uptake That
reaction
used for
generation
Still,
to the generation extensive
ATPase
preparation
the
been
transhydrogenase
to submitochondrial
essential
component
has already
when phospholipids
the transhydrogenase
as compared
an uncoupler-
potential.
of membrane
transhydrogenase
of stimulation
Since
generation
in general of the
agents
absence
catalyzes
of a membrane
to the
A stimulation
tution. in
the generation
in membranes
uncoupling
transhydrogenase
purified
of NAD+ by NADPH and an uncoupler-sensitive
to be related
coupling
communication
mitochondrial
reduction
appears
in this
of protons
(15). of ATP-driven
Vol. 67, No. 2, 1975
BIOCHEMICAL
transhydrogenase
using
transhydrogenase.
It
preparation
Complex
activity
of 4, probably
found
Complex
In contrast
demonstrated
in this
the
lipid
transhydrogenase,
to be optimal
reductase)
as the source
1,reconstituted (hydrophobic
to the paper,
a phosphatidylethanolamine
reflecting
RESEARCH COMMUNICATIONS
with protein)
values
authors
of the ATPase
found
in the reconstitution
a
of the Pi-ATP
ratio
system
the same phospholipid
of
with
to phosphatidylcholine
requirement since
these
a crude
reduction
optimal
of
and soybean
oligomycin-sensitive
by ATP.
with
of the
ATPase
an uncoupler-and
phosphatidylcholine
that
that
of oligomycin-sensitive
NADP' by NADH, driven
than
I (NADH-ubiquinone
was shown
phospholipida,catalyzed
maximal
AND BIOPHYSICAL
ratio
exchange
rather has been
(10).
ACKNOWLEDGEMENT This Biology Dr.
work
was carried
Organization
C. Miller
for
out during
fellowship his
advice
the
awarded regarding
tenure
of a European
to J.R.
The authors
Molecular wish
to thank
the TPB- measurements.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Rydstrzm, J., Hoek, J.B. and Ernster, L. (1975) The Enzymes, in press Racker, E. (1962) Proc. Natl. Acad. Sci. USA, 68, 1659-1663 Beyer, R.E. (1967) Methods in Enzymology I.& 186-194 Jacobs, E.E., Jacob, M., Sanadi, D.R. and Bradley, L.B. (1956) J. Biol. Chem. 223, 147-156. Lowry, O.H., Rosebrough,N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Hoek, J.B. and Hundal, T. (1974) Biochem.Biophys.Res.Commun. Rydstrom,J., 6CJ 448-455. Williams, J.N. (1964) Arch. Biochem. Biophys. 107, 537-543. Ragan, C.I. and Racker, E. (1973) J. Biol. Chem. 248, 2563-2569. Grinius,L.L., Jasaitis, A.A., Kadziauskas, Y.P., Liberman, E.A., Skulachev, V.P., Topali,V.P., Tsofina, L.M. and Vladimirova, M.A. (1970) Biochim. Biophys. Acta 216, l-12. Kagawa, Y., Kandrach, A. and,,Racker, E. (1973) J. Biol. Chem. 248, 676-684. Teixeira da Cruz, A., Rydstrom, J. and Ernster, L. (1971) Eur. J. Biochem. 3, 203-211. Skulachev, V.P. (1971) in Current Topics in Bioenergetics, ~01.4, Academic Press, New York and Lon‘;T;;n, p. 127-185. Asami, K., Juntti, K. and Ernster, L. (1970) Biochim. Biophys. Acta 205, 307-311. Ernster, L., Juntti, K. and Asami, K. (1972) Bioenergetics 5, 351-357. Mitchell, P. (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research,Bodmin. Ragan, C.I. and Widger, W.R. (1975) Biochem. Biophys. Res. Commun. 62, 744749.
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