Vol.
86,
February
No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
28, 1979
1248-1255
BOUND NUCLEOTIDES AND PHOSPHORYLATION IN RHODOSPIRILLUM RUBRUM David A. Harris* and Margareta Baltscheffsky Arrhenius Laboratory, Department of Biochemistry, University of Stockholm, S-106 91 Stockholm, Sweden
Received
January
4,1979 -3
SUhrnARY. Chromatophores from Rhodospirillum rubrum contain 12 x 10 mol ATP and 8.3 x 10-3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation. INTRODUCTION.
Coupling
bound ATP (2 mol/mol In 1973,
Harris
ATPase)
--et al-[21
in the mechanism
step
should
suggested
that
that
The essence
bound
and was thus out
ATPase).
these
being
The tightly
be pointed
of organisms (For
nucleotides of this
on the ATPase required
in phosphorylation
in phosphorylation,
variety
and ADP (lmol/mol
ATP synthesis
ii)
It
from a wide
of ATP synthesis. i)
requiring
ATPases
bound
the release
tightly
a review
were
directly
hypothesis
was:
no energy,
the
the
site
energy-
enzyme,
a (low-energy)
at an active
see [l]).
involved
of ATP from
ATP represented
no proposals
bear
intermediate
one of its
in
and
were made as to the role
states.
of the bound
ADP. The first
postulate,
supported
by work
formation,
is
NTP-driven
reactions
[5-7,
see also
to allow *
would
exchange 11.
assignment
Present
Abbreviations: opened ATP) ether.
Address.
of the with
However,
requirement
second added
the time
particles postulate, nucleotide resolution
for reaction
it only
was shown that under
of these
1248
bond and on
the bound
experiments
conditions was too
of phosphorylation.
Department of Biochemistry, University 9 Hyde Terrace, Leeds, Yorkshire, U.K.
@ I979 by Academic Press, Inc. in anyform reserved.
[3]
energised
of Leeds,
rro-ATP NTP, NDP, nucleoside tri, diphosphate. 2-2 [1-(9-adenyl)-l'-triphosphoryl-oxymethyl)-dihydroxydiethyl
of reproduction
ADP-Pi
[4].
of the bound ATP to the mechanism
0006-291X/79/041248-08$01.00/0 Copyrighr All rights
energy
on the Pi - H2180 exchange
in submitochondrial
In support nucleotide
of a nil
(ribose-ring
low
Attempts
Vol.
86,
No.
4, 1979
to
increase
of
an
inhibitor
fraction
of
Kinetic
studies
and is
the
resolution
ATP
not
an
show
what
and
indeed
that,
active
or
inactive
flash)
ATP
synthase
a single turnover
R.
rubrum,
would
formed It
is
bound It
was
thus
ATP
from
below
concluded
as
expected
not
the
shown
very
ATP
while
newly
one
or free
bound
lead
to
dADP, ATP
ATP
does
IDP not
is
number
Harris,
absent
order
to
chain
(by
a 10~s
all
coupled
ATP
produced,
of the
the
production
corresponding
below)
in
~101. nucleotides[
the
source dADP
of or
of
largely
([13]
adenine
turnovers
time
([lo],
measure
phosphorylated two
could
the
turnover
molecules
for
ATP
We have
respiratory
phosphorylation,
or
the
chloroplasts,
chloroplasts
specific
in
a similar
to
ATPase
also
I.111
publication)
a single
to
in
for
during
tightly
of
situation is
bound
released that
be
studies
protein
luciferase
cells
bound
protein for
of
the
cause
Using indeed
inhibitor
is
of
to
in
inhibitor
rate
turnover
a substrate
that,
ATPase
coli
that
over
a small
[ 9,101
a conclusion. as
the
only
of __E.
these
such
presence
flashes
imply
turning
the
allowing
ATP
to
submitted
G.K.,
fraction to
taken
draw
by
hydrolyses
luciferase
used
was
limited
present.
contrast
bound
particles
the
can
the
were
to
on
short
However,
valid
Radda,
a substantial
- whether shown
is
be
flash
of
is
not
during
been
molecules
A single
purified
dADP
is
dark
molecules
Since say,
the
rate.
in
ATPase
and
- e.g.
turnover
to
of
chromatophores,
phosphorylation light
phosphorylation.
molecules
rubrum
] have
COMMUNICATIONS
chloroplasts, [lo],
active
P into
submitochandrial
Tscharner:,V.
R.
[12
be
32
of
in
it
in
ATPase
van In
thus
in
RESEARCH
phosphorylation to
incorporation
fraction
shown
limits
molecules
intermediate
BIOPHYSICAL
foundered,
particles
measurement,
D.A.,
which
synthase on
AND
[8-lo]
protein
submitochondrial
not
of
BIOCHEMICAL
the
GDP were
occupy
an
141,
of
- can
the be
respiratory indeed active
first
if, NTP
determined. chain,
phosphorylated. site
for
phosphorylation. MATERIALS AND METHODS R. rubrum chromatophores were prepared by the method of Haltscheffsky [15]. Total phosphorylation was measured using a sensitive pH electrode, by the method of Nishimura et al [16]. Phosphorylation of ADP by the chromatophores was typically 8-9 molATP/mol chlorophyll/min under saturating light. Luciferase, purified by the method of Lundin [17] and luciferin were kindly supplied by Dr. A. Lundin. Luminescence was measured in an apparatus constructed
1249
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by B. HUijer in this laboratory. Actinic illumination was obtained via a light guide provided with double layers of Wratten 88A gelatin filter, and the photomultiplier tube was protected from scattered light with a Corning 9782 glass filter. Nucleotides were obtained as described elsewhere [4]. None of the triphosphates of those used here gave more than 5% of the luminescence observed with ATP at comparable concentrations in the luciferase assay (not shown). The bound nucleotide content of the chromatophore membrane was measured essentially as described by Harris and Slater [5].
RESULTS AND DISCUSSION. Table
I shows the
(as extracted
Bound
by 4% perchloric
of Ferguson
--et al.
[18]
acid).
with
The bound
adenine susceptible
This
I also
of added
and incomplete
phores
II
no other
I).
is
to a value
M.,
of
by the method
unpublished), sites
is close
extent
dATP also
chromatophores
estimated
binding
we see that
for
with
Also
to 1.5.
added
the bound
or
[19].
[ 14C]ATP
seems to be able
have a wide
to displace
ADP is much more
reversal
presented
on the coupling
ATPase
represents
transfer, NDP-kinase
that
solution.
and release
The release
is
a competition
slow
between
of (t
- 3s), 4 -. the
ATP.
processes.
specificity
active below
it
in the
of ATP to the
to submitochondrial
of electron
may be due to the very
release
illuminated
ATP (or phosphorylation
the released
nucleotide
are
are present.
because
in contrast
particles
is a slow
nucleotides
of coupled
shows that,
is
when washed
of bound
- presumably
specificity
NIP-driven
evidence
to a limited
there
release
and luciferase
Nucleotide Table
of ATPase
of antimycin
in particular,
nucleotide,
ADP) since
ATPase
correspond
and Baltscheffsky,
(Table
shows that,
must represent
bound
values
in R.rubrum
the ATP/ADP ratio
exchange
and,
bound
to displacement.
Table absence
amount
[l],
energised
nucleotides,
the D.A.
systems
nucleotides is
These
to the number
other
when the membrane
if
(Harris,
assumed to correspond
in agreement
in R. rubrum.
amount of ATP and ADP tightly
1 - 2 mol ATP per mol ATPase
is
nucleotides
of
particles
(coupled)
as well activity
phosphorylation
as of hydrolysis. in these
dADP, IDP and GDP, at least,
directly.
1250
[4],
particles.
chromatoand In part
this
However,
are phosphorylated
BIOCHEMICAL
Vol. 86, No. 4, 1979
TABLE I.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bound nucleotides'in
ATP bound
R. rubrum chromatophores [ 14 C]nucleotide ATP released on bound continuous illumination
ADP bound
(mmol per mol bacteriochlorophyll) [14c]~~p
12
8.3
dATP
10
2.3
2.2
4.3
1.0
Chromatophores were labelled with 200pM [14c]~~p under illumination as described [5] except that the buffer used contained 2OOmM glycylglycine, 5mM NaPi, 2mM MgC12 and O.lmM succinate (pH 7.4 with NaOH)14 Treatment with dATP was carried out similarly, with 2OOpM dATP replacing 1 C]ATP. Bound nucleotides were assayed, with luciferase, as described [5]. ATP release was measured by illuminating chromatophores with saturating, continuous light in the presence of luciferase + luciferin in the photometer described in 'Methods'. Control particles (prepared without preincubation) contained bound ATP and ADP identical in amount (within experimental error) to particles pre-incubated with [14c]~~p. Release of ATP on illumination was also identical (not shown).
TABLE II. Nucleoside
Nucleotide
base
of coupled
Phosphorylation
(no Pi)
processes
NADf reduction
1ooa 69 46 49 24 18
86,
February
No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
28, 1979
1248-1255
BOUND NUCLEOTIDES AND PHOSPHORYLATION IN RHODOSPIRILLUM RUBRUM David A. Harris* and Margareta Baltscheffsky Arrhenius Laboratory, Department of Biochemistry, University of Stockholm, S-106 91 Stockholm, Sweden
Received
January
4,1979 -3
SUhrnARY. Chromatophores from Rhodospirillum rubrum contain 12 x 10 mol ATP and 8.3 x 10-3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation. INTRODUCTION.
Coupling
bound ATP (2 mol/mol In 1973,
Harris
ATPase)
--et al-[21
in the mechanism
step
should
suggested
that
that
The essence
bound
and was thus out
ATPase).
these
being
The tightly
be pointed
of organisms (For
nucleotides of this
on the ATPase required
in phosphorylation
in phosphorylation,
variety
and ADP (lmol/mol
ATP synthesis
ii)
It
from a wide
of ATP synthesis. i)
requiring
ATPases
bound
the release
tightly
a review
were
directly
hypothesis
was:
no energy,
the
the
site
energy-
enzyme,
a (low-energy)
at an active
see [l]).
involved
of ATP from
ATP represented
no proposals
bear
intermediate
one of its
in
and
were made as to the role
states.
of the bound
ADP. The first
postulate,
supported
by work
formation,
is
NTP-driven
reactions
[5-7,
see also
to allow *
would
exchange 11.
assignment
Present
Abbreviations: opened ATP) ether.
Address.
of the with
However,
requirement
second added
the time
particles postulate, nucleotide resolution
for reaction
it only
was shown that under
of these
1248
bond and on
the bound
experiments
conditions was too
of phosphorylation.
Department of Biochemistry, University 9 Hyde Terrace, Leeds, Yorkshire, U.K.
@ I979 by Academic Press, Inc. in anyform reserved.
[3]
energised
of Leeds,
rro-ATP NTP, NDP, nucleoside tri, diphosphate. 2-2 [1-(9-adenyl)-l'-triphosphoryl-oxymethyl)-dihydroxydiethyl
of reproduction
ADP-Pi
[4].
of the bound ATP to the mechanism
0006-291X/79/041248-08$01.00/0 Copyrighr All rights
energy
on the Pi - H2180 exchange
in submitochondrial
In support nucleotide
of a nil
(ribose-ring
low
Attempts
Vol.
86,
No.
4, 1979
to
increase
of
an
inhibitor
fraction
of
Kinetic
studies
and is
the
resolution
ATP
not
an
show
what
and
indeed
that,
active
or
inactive
flash)
ATP
synthase
a single turnover
R.
rubrum,
would
formed It
is
bound It
was
thus
ATP
from
below
concluded
as
expected
not
the
shown
very
ATP
while
newly
one
or free
bound
lead
to
dADP, ATP
ATP
does
IDP not
is
number
Harris,
absent
order
to
chain
(by
a 10~s
all
coupled
ATP
produced,
of the
the
production
corresponding
below)
in
~101. nucleotides[
the
source dADP
of or
of
largely
([13]
adenine
turnovers
time
([lo],
measure
phosphorylated two
could
the
turnover
molecules
for
ATP
We have
respiratory
phosphorylation,
or
the
chloroplasts,
chloroplasts
specific
in
a similar
to
ATPase
also
I.111
publication)
a single
to
in
for
during
tightly
of
situation is
bound
released that
be
studies
protein
luciferase
cells
bound
protein for
of
the
cause
Using indeed
inhibitor
is
of
to
in
inhibitor
rate
turnover
a substrate
that,
ATPase
coli
that
over
a small
[ 9,101
a conclusion. as
the
only
of __E.
these
such
presence
flashes
imply
turning
the
allowing
ATP
to
submitted
G.K.,
fraction to
taken
draw
by
hydrolyses
luciferase
used
was
limited
present.
contrast
bound
particles
the
can
the
were
to
on
short
However,
valid
Radda,
a substantial
- whether shown
is
be
flash
of
is
not
during
been
molecules
A single
purified
dADP
is
dark
molecules
Since say,
the
rate.
in
ATPase
and
- e.g.
turnover
to
of
chromatophores,
phosphorylation light
phosphorylation.
molecules
rubrum
] have
COMMUNICATIONS
chloroplasts, [lo],
active
P into
submitochandrial
Tscharner:,V.
R.
[12
be
32
of
in
it
in
ATPase
van In
thus
in
RESEARCH
phosphorylation to
incorporation
fraction
shown
limits
molecules
intermediate
BIOPHYSICAL
foundered,
particles
measurement,
D.A.,
which
synthase on
AND
[8-lo]
protein
submitochondrial
not
of
BIOCHEMICAL
the
GDP were
occupy
an
141,
of
- can
the be
respiratory indeed active
first
if, NTP
determined. chain,
phosphorylated. site
for
phosphorylation. MATERIALS AND METHODS R. rubrum chromatophores were prepared by the method of Haltscheffsky [15]. Total phosphorylation was measured using a sensitive pH electrode, by the method of Nishimura et al [16]. Phosphorylation of ADP by the chromatophores was typically 8-9 molATP/mol chlorophyll/min under saturating light. Luciferase, purified by the method of Lundin [17] and luciferin were kindly supplied by Dr. A. Lundin. Luminescence was measured in an apparatus constructed
1249
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by B. HUijer in this laboratory. Actinic illumination was obtained via a light guide provided with double layers of Wratten 88A gelatin filter, and the photomultiplier tube was protected from scattered light with a Corning 9782 glass filter. Nucleotides were obtained as described elsewhere [4]. None of the triphosphates of those used here gave more than 5% of the luminescence observed with ATP at comparable concentrations in the luciferase assay (not shown). The bound nucleotide content of the chromatophore membrane was measured essentially as described by Harris and Slater [5].
RESULTS AND DISCUSSION. Table
I shows the
(as extracted
Bound
by 4% perchloric
of Ferguson
--et al.
[18]
acid).
with
The bound
adenine susceptible
This
I also
of added
and incomplete
phores
II
no other
I).
is
to a value
M.,
of
by the method
unpublished), sites
is close
extent
dATP also
chromatophores
estimated
binding
we see that
for
with
Also
to 1.5.
added
the bound
or
[19].
[ 14C]ATP
seems to be able
have a wide
to displace
ADP is much more
reversal
presented
on the coupling
ATPase
represents
transfer, NDP-kinase
that
solution.
and release
The release
is
a competition
slow
between
of (t
- 3s), 4 -. the
ATP.
processes.
specificity
active below
it
in the
of ATP to the
to submitochondrial
of electron
may be due to the very
release
illuminated
ATP (or phosphorylation
the released
nucleotide
are
are present.
because
in contrast
particles
is a slow
nucleotides
of coupled
shows that,
is
when washed
of bound
- presumably
specificity
NIP-driven
evidence
to a limited
there
release
and luciferase
Nucleotide Table
of ATPase
of antimycin
in particular,
nucleotide,
ADP) since
ATPase
correspond
and Baltscheffsky,
(Table
shows that,
must represent
bound
values
in R.rubrum
the ATP/ADP ratio
exchange
and,
bound
to displacement.
Table absence
amount
[l],
energised
nucleotides,
the D.A.
systems
nucleotides is
These
to the number
other
when the membrane
if
(Harris,
assumed to correspond
in agreement
in R. rubrum.
amount of ATP and ADP tightly
1 - 2 mol ATP per mol ATPase
is
nucleotides
of
particles
(coupled)
as well activity
phosphorylation
as of hydrolysis. in these
dADP, IDP and GDP, at least,
directly.
1250
[4],
particles.
chromatoand In part
this
However,
are phosphorylated
BIOCHEMICAL
Vol. 86, No. 4, 1979
TABLE I.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bound nucleotides'in
ATP bound
R. rubrum chromatophores [ 14 C]nucleotide ATP released on bound continuous illumination
ADP bound
(mmol per mol bacteriochlorophyll) [14c]~~p
12
8.3
dATP
10
2.3
2.2
4.3
1.0
Chromatophores were labelled with 200pM [14c]~~p under illumination as described [5] except that the buffer used contained 2OOmM glycylglycine, 5mM NaPi, 2mM MgC12 and O.lmM succinate (pH 7.4 with NaOH)14 Treatment with dATP was carried out similarly, with 2OOpM dATP replacing 1 C]ATP. Bound nucleotides were assayed, with luciferase, as described [5]. ATP release was measured by illuminating chromatophores with saturating, continuous light in the presence of luciferase + luciferin in the photometer described in 'Methods'. Control particles (prepared without preincubation) contained bound ATP and ADP identical in amount (within experimental error) to particles pre-incubated with [14c]~~p. Release of ATP on illumination was also identical (not shown).
TABLE II. Nucleoside
Nucleotide
base
of coupled
Phosphorylation
(no Pi)
processes
NADf reduction
1ooa 69 46 49 24 18
