BIOCHEMICAL
Vol. 78, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PURIFICATION
AND MOLECULAR PROPERTIES OF RECONSTITUTIVELY ACTIVE
NICOTINAMIDE
NUCLEOTIDE TRANSHYDROGENASEFRO?4BEEF HEART
MITOCHONDRIA. Bo Hijjeberg Department
of Biochemistry,
Stockholm,
S-106
Received
August
and Jan Rydstrijm Arrhenius
91 Stockholm, 29,
Laboratory,
University
of
Sweden.
1977
SUMMARY: Nicotinamide nucleotide transhydrogenase from beef heart The mitochondria was purified to homogeneity and characterized. enzyme is devoid of other respiratory chain activities as well as flavin. Reduction of NAD+ by NADPH catalyzed by reconstituted transhydrogenase generates an uncoupler-sensitive uptake of lipophilic anions, whereas the rate of reduction of NAD* by NADPH is enhanced about 13 fold by uncouplers. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate reveales that the protein consists of a single polypeptide of a molecular weight of 97,000. INTRODUCTION Nicotinamide has previously methods.
a definite there
one of the (see ref. fication
transhydrogenase
been partially
However,
allowed though
nucleotide
larger
that
components
of
a review).
is
transhydrogenase
for
the
is composed of
weight
of
about
97,000
enzyme
reconstituted
which
with
of
the enzyme,
present
a single
apparently
liposomes
inner
time.
tightly
It
is
shown that of a molecular
a flavoprotein. coupled
II-
Abbreviation: hydrazone.
FCCP, carbonyl
cyanide
the puri-
transhydrogenase
polypeptide is not
is
membrane
communication
mitochondrial first
al-
constitutes
the mitochondrial
active
reported
of purification
transhydrogenase
In the
of reconstitutively
to homogeneity
extent
as to the size
indications
heart
by a number of different
in no case has the conclusion
are
1 for
purified
from beef
p-trifluoromethoxyphenyl-
as indi-
The
Vol. 78, No. 4, 1977
cated philic catalytic
BIOCHEMICAL
by the generation anions
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of uncoupler-sensitive
and by a more than
activity
13 fold
uptake
stimulation
of
lipo-
of the
by uncouplers.
MATERIALS AND METHODS EDTA-submitochondrial particles were prepared as described Protein was determined by the biuret by Lee and Ernster (2). solutions, by the method (3), or, in the case of very dilute method of Bradford (4). Submitochondrial particles were fractionated with ammonium sulphate as described previously (5), except that the centrifugation steps were carried out with a Aliquots Beckman 35 rotor for 15 minutes at 90,000 x g (average). of 38-43P were chromatographed directly on DEAE-Sepharose CL-6B (column size 2 x 25 cm), equilibrated with 20 mM tricine (pH 8.0), 0.05 % Triton X-100, 3 mM EDTA and 30 % glycerol at a flow rate of 14-16 cm/h, after 3-fold dilution with equilibration buffer. After DEAE-Sepharose chromatography, active fractions were pooled, concentrated to 5 ml in an Amicon ultrafiltration cell (PM 10 filter, Amicon Corp., Lexington, Yass., USA) and made 10 mM with respect to potassium phosphate (with a 1 M potassium phosphate stock solution, pH 6.5). Hydroxylapatite chromatography (column size 1.5 x 3 cm) was performed after equilibration with 10 mM potassium phosphate (pH 6.5), 30 % glycerol and 0.05 % Triton x-100. The most active transhydrogenase was e1ute.l with 100 IW potassium phosphate. The preparation is about 50 % inactivated after 24 h at 4O, but is stable for at least a week when reconstituted with phospholipids and stored at the same temperature. Transhydrogenase activity was assayed at pH 7.4 as reduction of NAD+ by NADPH with lactate dehydrogenase as described previously (6), with 0.8 mM lysophosphatidylcholine added to the medium. The reaction was followed at 366-400 nm with an Aminco-Chance DW-2 spectrophotometer using a mmolar extinction coefficient of 3.1. NADH dehydrogenase (7), succinate dehydrogenase (8) and ATPase (9) were assayed as described earlier. Reconstitution and measurements of uptake of tetraphenylboron were carried out essentially according to Rydstrijm et al. (i'), except that synthetic dioleoyl-L-aphosphatidylcholineand 4 % cholate was used. Pure transhydrogenase (24 ug in 200 ~1 equilibration medium) was reconstituted with a presonicated concentrated cholate-lecithin suspension (in 100 ul) . Absorption spectra were recorded with a Cary model 17 spectFlavin content was determined according to Faeder rophotometer. and Siegel (10) with an Aminco Bowman spectrofluorometer. Polyacrylamide gel electrophoresis of transhydrogenase in the presence of sodium dodecyl sulphate was carried out on a high resolution gradient (7.5-15 %) slab polyacrylamide gel according to Alvares and Siekevitz (11). Bovine serum albumine (monomer and diner), catalase, ovalbumine and trypsin inhibitor were employed as reDEAE-Sepharose CL-6B was purchased from Pharference proteins. macia Fine Chemicals AB, Uppsala, Sweden. Hydroxylapatite (Bio-Gel HTP), acrylamide and bis(N,N '-methylene-bis-acrylamide were obtained from Bio-Rad Laboratories, Richmond, Ca., USA. Lysophosphatidylcholine (egg), dioleoyl-L-a-phosphatidylcholine and other biochemicals were obtained from Sigma Chem. Co., St. Louis, Miss., USA.
1184
Vol. 78, No. 4, 1977
BlOCHEMlCAl
AND BlOPHYSlCAl
Table Purification
1
of transhydrogenase submitochondrial
Total protein (mg)
RESEARCH COMMUNICATIONS
from beef
heart
particles
Total activity (units)
Specific activity (units/mg)l
Purification (x-fold)
Submitochondrial particles
85
9.52
0.11
1
38-43P
23
5.19
0.25
2.3 8.2
DEAE-Sepharose pool
1.7
1.53
0.90
Hydroxylapatite pool
0.19
0.83
4.4
1 1 unit is defined as a transhydrogenase NADH formed per minute.
40
activity
of
1 umole
RESULTS Transhydroqenase to DEAE-Sepharose buffer,
CL-6B.
After
from 0 to
beginning
of
substantial in the i.e.,
0.4 PI.
the gradient
part
of
cytochromes, still
higher
activates zyme is
is retained salt
not retained
concentrated (cf. -
fractions
and a is
eluted
some red material,
on the column and can be eluted
Also,
on the column
Too extensive it
is
important
longer
than
and applied
Transhydrogenase
1185
washing that
the
necessary.
(minimum 0.5 umoles/min/mg) cell
KC1
in the very
flavins,
whereas
binds
a linear
0.1 M KCl)
i.e.,
concentrations.
in an Amicon Methods).
with
is eluted
approximately material,
Methods)
30 ml equilibration
is eluted
the gradient,
transhydrogenase.
most active
column
(at
(cf. -
with
Transhydrogenase
amount of yellow
latter
38-43P
washing
the bound transhydrogenase
gradient
with
of the diluted
enThe
are pooled,
to a hydroxylapatite
eluted
with
in-
100 mM po-
Vol. 78, No. 4, 1977
BlOCHEMlCAL
0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5
10 MOBILITY
Fig.
1.
phosphate
between
2.5 and 5.8 ~moles/min/mg
about
10 %.
Table
1.
(cf. -
with
succinate
Methods)
The complete
The final
associated
preparation
that
flavin.
allows
that
A direct
The sensitivity
Polyacrylamide
that
bound flavin gel
about
97,000
daltons.
spectrum
most likely flavin
(cf. -
is accounted
less
The size
of
transhydrogenase
(Fig.
1) shows that
this
1186
Methods) amounts
than
by a single
0.1 mole
subunit.
of
for
is not
detectable
per mole transhydrogenase
sulphate
bet-
of the method thus
enzyme contains
electrophoresis
sence of sodium dodecyl 100 % of the protein
the
activities
peak at 275 nm
does not contain and specificity
in
NADH dehydrogenase,
one absorption
of
is
is summarized
absorption
determination
yield
of various
e.g., Its
varying
the overall
transhydrogenase
transhydrogenase
the conclusion
noncovalently
chain,
only
activity
procedure
and ATPase.
shown) indicating
revealed
protein;
is devoid
the respiratory
dehydrogenase
a flavoprotein.
has a specific
purification
ween 250 and 600 nm reveals
of
20
Polyacrylamide gel electrophoresis of purified transhydrogenase in the presence of sodium dodecyl sulphate. Densitometric scanning at 560 nm of a slab gel containing 7 pg of protein stained with Coomassie Brilliant Blue G-250.
tassium
(not
15 Icml
in the preclose
to
polypeptide
band correlates
directly
of
Vol. 78, No. 4, 1977
A
BIOCHEMICAL
C
B
NADPH
NADPH
-4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
NADPH
-1
-4 f
C recons,.
B
submtl part
reconst 38-43P
-..A
pure --‘,,
-+ \
FCCP 4
transh
FCCP
FCCP 1
\
u t FCCP
t 1omv L
0
t FCCP
+ Zmwl-
2
t
i
\
c
AA=0005 1
03
+lmtn-
Fig.
2.
Uptake of tetraphenylboron catalyzed by submitochondrial particles (A), reconstituted partially purified (38-43P) transhydrogenase (B) and reconstituted pure transhydroyenase (C). Reconstitution and assay were carried out as described in Methods except that lysolecithin was omitted. Yedium contained the complete reaction mixture except NADPH. The amount of protein in each assay was: (A),100 pg; (B) , 80 p4; and (C),0.5 pg. The addition was 1 pg FCCP.
Fig.
3
Effect of FCCP on the rate of reduction of NADf by NADPH catalyzed by submitochondrial particles (submit. part., A), reconstituted partially purified transhydrogenase (reconst. 38-43P, B) and reconstituted pure transhydroqenase (reconst. pure transh., C). Reconstitution and assay were carried out as described in Vethods. The amount-or protein in each assay was: The addition (A) , 43 114; (B), 15 Vq; and (Cl, 1 pg. was 1 Vg FCCP.
to
the
specific
purification 97,000 ive
is
of
the transhydrogenase
procedure.
It
may therefore
the minimal
partially
weight
to submitochondrial
purified an NAD+ plus
tetraphenylboron 5) -
molecular
throughout
be concluded
the
that
of the catalytically
act-
transhydrogenase. Similar
lyzes
activity
The rate
transhydrogenase, NADPH dependent (Fig.
of
particles
pure transhydrogenase uncoupler-sensitive
2) when reconstituted
reduction
and reconstituted
with
uptake liposomes
of NAD+ by NADPH catalyzed
1187
cataof (cf. -
by the re-
Vol. 78, No. 4, 1977
constituted
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pure transhydrogenase
by uncouplers
(Fig.
3).
This
submitochondrial
particles
transhydroqenase
which
see also
ref.
niqericin
5).
agents,
coupled
rate
is a considerable
Uncouplers
e.g.,
partially
of potassium
is not enhanced
further
(Fig.
by valinomycin
chloride
(not
over
purified
of 50 % or less
may be replaced
lysolecithin
13 fold
improvement
and reconstituted
show a stimulation
in the presence
rupting
is enhanced more than
3,
plus
or membrane-dis-
shown).
However,
by lysolecithin
the un-
(not
shown).
DISCUSSION The present minimal
paper demonstrates
molecular
transhydrogenase
weight
of beef
is about
97,000
not a flavoprotein. was apparently preparation
(3).
highly involved
between
and specific
unlikely
that
in the
shown whether
of
of
activity another
of
97,000
native
is
properties
component. the more likely of the
enzyme
Reconstitution
of
of submitochondrial nucleotide-binding surrounding
it
the pure
of
molecular
weight
it
appears
would be
remains
mitochondrial
of
the
the preparation
It
to be
transhydro-
that
the
units
latter
the complex
transhydrogenase liposomes
particles.
medium (cf. -
purity
peptide
appears
is
of
alter-
allosteric
(1).
of which
sites
the
weight
composed of multiple
one in view
dioleoyl-l-a-phosphatidylcholine enzyme the properties
low
of
the 97,000
reaction.
However,
the molecular
due to the
active
that
nucleotide
transhydrogenase,
qenase is a monomer or an oligomer the
of
or additional
catalytically
time
the enzyme probably
the homogeneity size
first
nicotinamide
estimate
transhydrogenase
the
the
and that
presumably
In view
and the correlation component
heart
A previous incorrect
for
appear
to be very
In both
systems
of
transhydrogenase
ref.
1) as indicated
1188
gives
with
synthetic
a membrane-bound similar
to those
the nicotinamide
are exposed
to the
by the
of sti-
lack
Vol. 78, No. 4, 1977
mulation
of
BIOCHEMICAL
the activity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of uncoupled
genase by membrane disruption.
lit
However, ed
indicating
anions,
a reduction
stimulated
by uncouplers
properties
of
liposomes fined energy
for
membrane it
appears
known polypeptide purification
(cf. -
indicates
reconstitut-
studies
is strongly
agents.
These
indicate
a simple
that
the
and well
de-
the mechanism of
on
about
0.1
A dimeric
structural
data
rogenase
(for
likely
the
(NADH-ubiquinone
transhydrogenase estimated.
reductase)
Whether
(17) because
structurally However,
it
molecular (16)
although
of circumstantial
linked
but
lower
times
about give
2 %
a value
than
those
intact
functional
indeed its
1189
component identical
However, of Complex to the
weight
was under-
fractionates
with
Complex I
or because
membrane is
possibility.
1).
molecular
reasons
the accumulating
is
or
and transhyd-
see ref.
weight
to NADH dehydrogenase
seems that
50
of NADH-dehydro-
a direct
problem
transhydrogenase
in Complex I or in the
those
(14,15)
largest
constitutes
NADH dehydrogenase
of this
87,000
subunit
to
to eliminate
between
a discussion that
the
inner
(14,15).
appear
relationship
represents
enzyme would thus
is close
dehydrogenase
of ATPase and cytochromes The present
in the mitochondrial
transhydrogenase
nmoles/mg which
genase and succinate
located
A maximum of approximately
that
of
former
providing
transhydrogenase 13).
the membrane protein.
I
further
proteins
that
of
is
of lipophi-
the
transhydrogenase thus
an
transduction. Compared to other
it
particles
or membrane-disrupting
coupled
suitable
system,
uptake
of NAD+ by NADPH that
the reconstituted
are tightly
catalyze
of a membrane potential.
to submitochondrial
catalyzes
transhydro-
systems
NADPH dependent
the generation
in contrast
system
both
Also,
SAD+ plus
uncoupler-sensitive
reconstituted
transhydrogenase
functionally remains
information
and/or
to be shown. favours
the
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENT We wish expert
technical
to thank
Mr.
Krister
Blomberg
and Mr.
Bo Hijijer
for
assistance.
This work was supported
by The Swedish Cancer
Society.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Rydstrom, J. (1977) Biochim. Biophys. Acta, in press. Lee, C.P. and Ernster, L. (1967) 14ethods in Enzymology 10, 543-548. Jacobs, E.E., Jacob, M., Sanadi, D.R. and Bradley, L.B. (1956) J. Biol. Chem. 223, 147-156. Bradford, M.M. 11976) Anal. Biochem. 2, 248-254. Rydstrom, J., Kanner, B. and Racker, E. (1975) Biochem. Biophys. Res. Commun. 67, 831-839. Teixeira da Cruz, A., Rydstrijm, J. and Ernster, L. (1971) Eur. J. Biochem. 2, 203-211. flinakami, S., Ringler, R.L. and Singer, T.P. (1962) J. Biol. Chem. 237, 569-576. King, T.E. (1963) J. Biol. Chem. 238, 4032-4036. Pullman, M.E., Penefsky, H.S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322-3329. Faeder, E.J. and Siegel, L.M. (1973) Anal. Biochem. 53, 332-336. P. (1973) Biochem. Biophys. Res. Alvares, A.P. and Siekevitz, Commun. 54, 923-929. (1966) Chemiosmotic Coupling in Oxidative and MitchellFP. Photosynthetic Phosphorylation, Glynn Research, Bodmin. J.W. and Ernster, L. (1977) Ann. Rev. Biochem. 46, DePierre, 201-262. Oxidations (T.P. Singer, Klingenberg, M. (1968) in Biological ed.) Wiley, New York, pp. 3-54. Chance, B., Azzi, A., Lee, I.Y., Lee, C.P. and Mela, L. (1969) and Function (L. Ernster and in "litochondria - Structure eds) Academic Press, London, pp. 233-273. Z. Drahota, (1976) Biochem. J. 154, 295-305. Ragan, C.I. Hatefi, Y. and Hanstein, W.G. (1973) Biochemistry -12, 35153522.
1190
Vol. 78, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PURIFICATION
AND MOLECULAR PROPERTIES OF RECONSTITUTIVELY ACTIVE
NICOTINAMIDE
NUCLEOTIDE TRANSHYDROGENASEFRO?4BEEF HEART
MITOCHONDRIA. Bo Hijjeberg Department
of Biochemistry,
Stockholm,
S-106
Received
August
and Jan Rydstrijm Arrhenius
91 Stockholm, 29,
Laboratory,
University
of
Sweden.
1977
SUMMARY: Nicotinamide nucleotide transhydrogenase from beef heart The mitochondria was purified to homogeneity and characterized. enzyme is devoid of other respiratory chain activities as well as flavin. Reduction of NAD+ by NADPH catalyzed by reconstituted transhydrogenase generates an uncoupler-sensitive uptake of lipophilic anions, whereas the rate of reduction of NAD* by NADPH is enhanced about 13 fold by uncouplers. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate reveales that the protein consists of a single polypeptide of a molecular weight of 97,000. INTRODUCTION Nicotinamide has previously methods.
a definite there
one of the (see ref. fication
transhydrogenase
been partially
However,
allowed though
nucleotide
larger
that
components
of
a review).
is
transhydrogenase
for
the
is composed of
weight
of
about
97,000
enzyme
reconstituted
which
with
of
the enzyme,
present
a single
apparently
liposomes
inner
time.
tightly
It
is
shown that of a molecular
a flavoprotein. coupled
II-
Abbreviation: hydrazone.
FCCP, carbonyl
cyanide
the puri-
transhydrogenase
polypeptide is not
is
membrane
communication
mitochondrial first
al-
constitutes
the mitochondrial
active
reported
of purification
transhydrogenase
In the
of reconstitutively
to homogeneity
extent
as to the size
indications
heart
by a number of different
in no case has the conclusion
are
1 for
purified
from beef
p-trifluoromethoxyphenyl-
as indi-
The
Vol. 78, No. 4, 1977
cated philic catalytic
BIOCHEMICAL
by the generation anions
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of uncoupler-sensitive
and by a more than
activity
13 fold
uptake
stimulation
of
lipo-
of the
by uncouplers.
MATERIALS AND METHODS EDTA-submitochondrial particles were prepared as described Protein was determined by the biuret by Lee and Ernster (2). solutions, by the method (3), or, in the case of very dilute method of Bradford (4). Submitochondrial particles were fractionated with ammonium sulphate as described previously (5), except that the centrifugation steps were carried out with a Aliquots Beckman 35 rotor for 15 minutes at 90,000 x g (average). of 38-43P were chromatographed directly on DEAE-Sepharose CL-6B (column size 2 x 25 cm), equilibrated with 20 mM tricine (pH 8.0), 0.05 % Triton X-100, 3 mM EDTA and 30 % glycerol at a flow rate of 14-16 cm/h, after 3-fold dilution with equilibration buffer. After DEAE-Sepharose chromatography, active fractions were pooled, concentrated to 5 ml in an Amicon ultrafiltration cell (PM 10 filter, Amicon Corp., Lexington, Yass., USA) and made 10 mM with respect to potassium phosphate (with a 1 M potassium phosphate stock solution, pH 6.5). Hydroxylapatite chromatography (column size 1.5 x 3 cm) was performed after equilibration with 10 mM potassium phosphate (pH 6.5), 30 % glycerol and 0.05 % Triton x-100. The most active transhydrogenase was e1ute.l with 100 IW potassium phosphate. The preparation is about 50 % inactivated after 24 h at 4O, but is stable for at least a week when reconstituted with phospholipids and stored at the same temperature. Transhydrogenase activity was assayed at pH 7.4 as reduction of NAD+ by NADPH with lactate dehydrogenase as described previously (6), with 0.8 mM lysophosphatidylcholine added to the medium. The reaction was followed at 366-400 nm with an Aminco-Chance DW-2 spectrophotometer using a mmolar extinction coefficient of 3.1. NADH dehydrogenase (7), succinate dehydrogenase (8) and ATPase (9) were assayed as described earlier. Reconstitution and measurements of uptake of tetraphenylboron were carried out essentially according to Rydstrijm et al. (i'), except that synthetic dioleoyl-L-aphosphatidylcholineand 4 % cholate was used. Pure transhydrogenase (24 ug in 200 ~1 equilibration medium) was reconstituted with a presonicated concentrated cholate-lecithin suspension (in 100 ul) . Absorption spectra were recorded with a Cary model 17 spectFlavin content was determined according to Faeder rophotometer. and Siegel (10) with an Aminco Bowman spectrofluorometer. Polyacrylamide gel electrophoresis of transhydrogenase in the presence of sodium dodecyl sulphate was carried out on a high resolution gradient (7.5-15 %) slab polyacrylamide gel according to Alvares and Siekevitz (11). Bovine serum albumine (monomer and diner), catalase, ovalbumine and trypsin inhibitor were employed as reDEAE-Sepharose CL-6B was purchased from Pharference proteins. macia Fine Chemicals AB, Uppsala, Sweden. Hydroxylapatite (Bio-Gel HTP), acrylamide and bis(N,N '-methylene-bis-acrylamide were obtained from Bio-Rad Laboratories, Richmond, Ca., USA. Lysophosphatidylcholine (egg), dioleoyl-L-a-phosphatidylcholine and other biochemicals were obtained from Sigma Chem. Co., St. Louis, Miss., USA.
1184
Vol. 78, No. 4, 1977
BlOCHEMlCAl
AND BlOPHYSlCAl
Table Purification
1
of transhydrogenase submitochondrial
Total protein (mg)
RESEARCH COMMUNICATIONS
from beef
heart
particles
Total activity (units)
Specific activity (units/mg)l
Purification (x-fold)
Submitochondrial particles
85
9.52
0.11
1
38-43P
23
5.19
0.25
2.3 8.2
DEAE-Sepharose pool
1.7
1.53
0.90
Hydroxylapatite pool
0.19
0.83
4.4
1 1 unit is defined as a transhydrogenase NADH formed per minute.
40
activity
of
1 umole
RESULTS Transhydroqenase to DEAE-Sepharose buffer,
CL-6B.
After
from 0 to
beginning
of
substantial in the i.e.,
0.4 PI.
the gradient
part
of
cytochromes, still
higher
activates zyme is
is retained salt
not retained
concentrated (cf. -
fractions
and a is
eluted
some red material,
on the column and can be eluted
Also,
on the column
Too extensive it
is
important
longer
than
and applied
Transhydrogenase
1185
washing that
the
necessary.
(minimum 0.5 umoles/min/mg) cell
KC1
in the very
flavins,
whereas
binds
a linear
0.1 M KCl)
i.e.,
concentrations.
in an Amicon Methods).
with
is eluted
approximately material,
Methods)
30 ml equilibration
is eluted
the gradient,
transhydrogenase.
most active
column
(at
(cf. -
with
Transhydrogenase
amount of yellow
latter
38-43P
washing
the bound transhydrogenase
gradient
with
of the diluted
enThe
are pooled,
to a hydroxylapatite
eluted
with
in-
100 mM po-
Vol. 78, No. 4, 1977
BlOCHEMlCAL
0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5
10 MOBILITY
Fig.
1.
phosphate
between
2.5 and 5.8 ~moles/min/mg
about
10 %.
Table
1.
(cf. -
with
succinate
Methods)
The complete
The final
associated
preparation
that
flavin.
allows
that
A direct
The sensitivity
Polyacrylamide
that
bound flavin gel
about
97,000
daltons.
spectrum
most likely flavin
(cf. -
is accounted
less
The size
of
transhydrogenase
(Fig.
1) shows that
this
1186
Methods) amounts
than
by a single
0.1 mole
subunit.
of
for
is not
detectable
per mole transhydrogenase
sulphate
bet-
of the method thus
enzyme contains
electrophoresis
sence of sodium dodecyl 100 % of the protein
the
activities
peak at 275 nm
does not contain and specificity
in
NADH dehydrogenase,
one absorption
of
is
is summarized
absorption
determination
yield
of various
e.g., Its
varying
the overall
transhydrogenase
transhydrogenase
the conclusion
noncovalently
chain,
only
activity
procedure
and ATPase.
shown) indicating
revealed
protein;
is devoid
the respiratory
dehydrogenase
a flavoprotein.
has a specific
purification
ween 250 and 600 nm reveals
of
20
Polyacrylamide gel electrophoresis of purified transhydrogenase in the presence of sodium dodecyl sulphate. Densitometric scanning at 560 nm of a slab gel containing 7 pg of protein stained with Coomassie Brilliant Blue G-250.
tassium
(not
15 Icml
in the preclose
to
polypeptide
band correlates
directly
of
Vol. 78, No. 4, 1977
A
BIOCHEMICAL
C
B
NADPH
NADPH
-4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
NADPH
-1
-4 f
C recons,.
B
submtl part
reconst 38-43P
-..A
pure --‘,,
-+ \
FCCP 4
transh
FCCP
FCCP 1
\
u t FCCP
t 1omv L
0
t FCCP
+ Zmwl-
2
t
i
\
c
AA=0005 1
03
+lmtn-
Fig.
2.
Uptake of tetraphenylboron catalyzed by submitochondrial particles (A), reconstituted partially purified (38-43P) transhydrogenase (B) and reconstituted pure transhydroyenase (C). Reconstitution and assay were carried out as described in Methods except that lysolecithin was omitted. Yedium contained the complete reaction mixture except NADPH. The amount of protein in each assay was: (A),100 pg; (B) , 80 p4; and (C),0.5 pg. The addition was 1 pg FCCP.
Fig.
3
Effect of FCCP on the rate of reduction of NADf by NADPH catalyzed by submitochondrial particles (submit. part., A), reconstituted partially purified transhydrogenase (reconst. 38-43P, B) and reconstituted pure transhydroqenase (reconst. pure transh., C). Reconstitution and assay were carried out as described in Vethods. The amount-or protein in each assay was: The addition (A) , 43 114; (B), 15 Vq; and (Cl, 1 pg. was 1 Vg FCCP.
to
the
specific
purification 97,000 ive
is
of
the transhydrogenase
procedure.
It
may therefore
the minimal
partially
weight
to submitochondrial
purified an NAD+ plus
tetraphenylboron 5) -
molecular
throughout
be concluded
the
that
of the catalytically
act-
transhydrogenase. Similar
lyzes
activity
The rate
transhydrogenase, NADPH dependent (Fig.
of
particles
pure transhydrogenase uncoupler-sensitive
2) when reconstituted
reduction
and reconstituted
with
uptake liposomes
of NAD+ by NADPH catalyzed
1187
cataof (cf. -
by the re-
Vol. 78, No. 4, 1977
constituted
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pure transhydrogenase
by uncouplers
(Fig.
3).
This
submitochondrial
particles
transhydroqenase
which
see also
ref.
niqericin
5).
agents,
coupled
rate
is a considerable
Uncouplers
e.g.,
partially
of potassium
is not enhanced
further
(Fig.
by valinomycin
chloride
(not
over
purified
of 50 % or less
may be replaced
lysolecithin
13 fold
improvement
and reconstituted
show a stimulation
in the presence
rupting
is enhanced more than
3,
plus
or membrane-dis-
shown).
However,
by lysolecithin
the un-
(not
shown).
DISCUSSION The present minimal
paper demonstrates
molecular
transhydrogenase
weight
of beef
is about
97,000
not a flavoprotein. was apparently preparation
(3).
highly involved
between
and specific
unlikely
that
in the
shown whether
of
of
activity another
of
97,000
native
is
properties
component. the more likely of the
enzyme
Reconstitution
of
of submitochondrial nucleotide-binding surrounding
it
the pure
of
molecular
weight
it
appears
would be
remains
mitochondrial
of
the
the preparation
It
to be
transhydro-
that
the
units
latter
the complex
transhydrogenase liposomes
particles.
medium (cf. -
purity
peptide
appears
is
of
alter-
allosteric
(1).
of which
sites
the
weight
composed of multiple
one in view
dioleoyl-l-a-phosphatidylcholine enzyme the properties
low
of
the 97,000
reaction.
However,
the molecular
due to the
active
that
nucleotide
transhydrogenase,
qenase is a monomer or an oligomer the
of
or additional
catalytically
time
the enzyme probably
the homogeneity size
first
nicotinamide
estimate
transhydrogenase
the
the
and that
presumably
In view
and the correlation component
heart
A previous incorrect
for
appear
to be very
In both
systems
of
transhydrogenase
ref.
1) as indicated
1188
gives
with
synthetic
a membrane-bound similar
to those
the nicotinamide
are exposed
to the
by the
of sti-
lack
Vol. 78, No. 4, 1977
mulation
of
BIOCHEMICAL
the activity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of uncoupled
genase by membrane disruption.
lit
However, ed
indicating
anions,
a reduction
stimulated
by uncouplers
properties
of
liposomes fined energy
for
membrane it
appears
known polypeptide purification
(cf. -
indicates
reconstitut-
studies
is strongly
agents.
These
indicate
a simple
that
the
and well
de-
the mechanism of
on
about
0.1
A dimeric
structural
data
rogenase
(for
likely
the
(NADH-ubiquinone
transhydrogenase estimated.
reductase)
Whether
(17) because
structurally However,
it
molecular (16)
although
of circumstantial
linked
but
lower
times
about give
2 %
a value
than
those
intact
functional
indeed its
1189
component identical
However, of Complex to the
weight
was under-
fractionates
with
Complex I
or because
membrane is
possibility.
1).
molecular
reasons
the accumulating
is
or
and transhyd-
see ref.
weight
to NADH dehydrogenase
seems that
50
of NADH-dehydro-
a direct
problem
transhydrogenase
in Complex I or in the
those
(14,15)
largest
constitutes
NADH dehydrogenase
of this
87,000
subunit
to
to eliminate
between
a discussion that
the
inner
(14,15).
appear
relationship
represents
enzyme would thus
is close
dehydrogenase
of ATPase and cytochromes The present
in the mitochondrial
transhydrogenase
nmoles/mg which
genase and succinate
located
A maximum of approximately
that
of
former
providing
transhydrogenase 13).
the membrane protein.
I
further
proteins
that
of
is
of lipophi-
the
transhydrogenase thus
an
transduction. Compared to other
it
particles
or membrane-disrupting
coupled
suitable
system,
uptake
of NAD+ by NADPH that
the reconstituted
are tightly
catalyze
of a membrane potential.
to submitochondrial
catalyzes
transhydro-
systems
NADPH dependent
the generation
in contrast
system
both
Also,
SAD+ plus
uncoupler-sensitive
reconstituted
transhydrogenase
functionally remains
information
and/or
to be shown. favours
the
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGEMENT We wish expert
technical
to thank
Mr.
Krister
Blomberg
and Mr.
Bo Hijijer
for
assistance.
This work was supported
by The Swedish Cancer
Society.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Rydstrom, J. (1977) Biochim. Biophys. Acta, in press. Lee, C.P. and Ernster, L. (1967) 14ethods in Enzymology 10, 543-548. Jacobs, E.E., Jacob, M., Sanadi, D.R. and Bradley, L.B. (1956) J. Biol. Chem. 223, 147-156. Bradford, M.M. 11976) Anal. Biochem. 2, 248-254. Rydstrom, J., Kanner, B. and Racker, E. (1975) Biochem. Biophys. Res. Commun. 67, 831-839. Teixeira da Cruz, A., Rydstrijm, J. and Ernster, L. (1971) Eur. J. Biochem. 2, 203-211. flinakami, S., Ringler, R.L. and Singer, T.P. (1962) J. Biol. Chem. 237, 569-576. King, T.E. (1963) J. Biol. Chem. 238, 4032-4036. Pullman, M.E., Penefsky, H.S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322-3329. Faeder, E.J. and Siegel, L.M. (1973) Anal. Biochem. 53, 332-336. P. (1973) Biochem. Biophys. Res. Alvares, A.P. and Siekevitz, Commun. 54, 923-929. (1966) Chemiosmotic Coupling in Oxidative and MitchellFP. Photosynthetic Phosphorylation, Glynn Research, Bodmin. J.W. and Ernster, L. (1977) Ann. Rev. Biochem. 46, DePierre, 201-262. Oxidations (T.P. Singer, Klingenberg, M. (1968) in Biological ed.) Wiley, New York, pp. 3-54. Chance, B., Azzi, A., Lee, I.Y., Lee, C.P. and Mela, L. (1969) and Function (L. Ernster and in "litochondria - Structure eds) Academic Press, London, pp. 233-273. Z. Drahota, (1976) Biochem. J. 154, 295-305. Ragan, C.I. Hatefi, Y. and Hanstein, W.G. (1973) Biochemistry -12, 35153522.
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