Biochem. J. (1972) 129, 471-481
Printed in Great Britain
471
Studies on the Light-Dependent Synthesis of Inorganic Pyrophosphate
by Rhodospirillum rubrum Chromatophores
By RICHARD J. GUILLORY
Department ofBiochemistry and Biophysics,
University of Hawaii, Honolulu, Hawaii 96822, U.S.A.
and
RONALD R. FISHER
Department of Chemistry
University of South Carolina, Columbia, S.C. 29208, U.S.A.
(Received 10 March 1972)
Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate
were examined in chromatophores of Rhodospirillum rubrum. The application of an
ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion
that pyrophosphate is a product of a light-dependent reaction utilizing Pi as the substrate.
Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis
of both ATP and pyrophosphate were studied under conditions in which the membranebound adenosine triphosphatase and pyrophosphatase activities would normally make
these substances unstable. The maximum rate of pyrophosphate synthesis was 25 % ofthat
for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a
lower light-intensity than that required for ATP synthesis. As a result, at low lightintensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of
synthesis of both pyrophosphate and ATP were attained only on the addition of an
exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required
about one-half of the concentration ofthe reductant required for maximum ATP synthesis.
Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little
influence on the rate of pyrophosphate synthesis. In membrane particles that retained
pyrophosphatase activity but were treated to remove adenosine triphosphatase activity
and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent
pyrophosphate synthesis by nearly 250 %. The influence of Mg2+ concentration, pH and
various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results
are discussed with respect to the mechanism and function of electron-transport-coupled
energy conservation in R. rubrum chromatophores.
Within the past 25 years PP1 has been shown to be
involved in the biosynthesis and transformation of a
wide variety of biological substances (Schmidt, 1961;
Kornberg, 1957). Many enzyme-catalysed reactions
occurring in subcellular particles produce PPi as one
of their reaction products; for example, ATP hydrolysis in microsomal preparations (Kennery et al.,
1957) and fatty acid activation in mitochondria
(Mahler, 1952). In addition, PP1 is formed during
protein synthesis at the stage of amino acid activation
(Hoagland et al., 1956), during nucleic acid synthesis
at the polymerization step (Kornberg, 1960) and in
polysaccharide synthesis and saccharide interconversion in the formation of the glycosyl donor (Leloir
& Cardini, 1957). The pyrophosphatase activity of
many cells and subcellular particles may be responsible for directing biosynthetic reaction by hydrolysis
of the PPi product (Kornberg, 1962).
Vol. 129
Model systems which couple the endergonic formation of PP1 from Pi to exergonic oxidation-reduction
reactions have been reported (Clark et al., 1958;
Baltrop, 1963). That such model synthesis could have
its energy-conserving biological counterpart was first
suggested by Calvin (1963). This suggestion was
supported by the later discovery of a light-dependent
disappearance of Pi in Rhodospirillum rubrum chromatophores, in which the product of the reaction was
specified as being PPi (Baltscheffsky & von Stedingk,
1966; Baltscheffsky et al., 1966).
The reversibility of the chromatophore PPI-synthesizing system was indicated by the findings that
PP1 functions as an energy donor for the energy-linked
nicotinamide nucleotide transhydrogenase (Keister &
Yike, 1967a), for the reduction of NADI by several
electron donors of high oxidation-reduction potential
(Keister & Yike, 1967b) and for the reversal of
R. J. GUILLORY AND R. R. FISHER
472
electron transport, indicated by the apparent reduction of a membrane-bound b-type cytochrome
(Baltscheffsky, 1968).
We have prepared R. rubrum chromatophore
particles totally deficient in ATPase* or pyrophosphatase activity (Fisher & Guillory, 1969a,c). With
these particles the terminal phosphorylation reactions
leading to synthesis of PPi and ATP were demonstrated to be catalysed by two distinct enzymes. In
the present paper we describe some of the characteristics of synthesis of PP1 and ATP in intact and
in modified chromatophores of R. rubrum. An ADPglucose pyrophosphorylase-trapping system is utilized, which permits the unambiguous, sensitive and
quantitative evaluation of PP1 synthesis (Guillory &
Fisher, 1971). The trapping system has been used in
the investigation of the rates of formation of ATP
and PP1 under conditions where both synthetic
reactions can occur simultaneously.
Materials and Methods
Glucose 6-phosphate dehydrogenase (EC 1.1.1.49),
hexokinase (EC 2.7.1.1), phosphoglucomutase (EC
2.7.5.1) and pyruvate kinase (EC 2.7.1.40) were obtained from Boehringer (Mannheim) Corp., New
York, N.Y., U.S.A.; ADP-glucose, ADP, x-D-glucose 1-phosphate, oc-D-glucose 6-phosphate, NADP+,
NAD+, NADH, protamine sulphate (grade I), glycylglycine and oligomycin were from Sigma Chemical
Co., St. Louis, Mo., U.S.A. All other reagents were
of analytical grade and were obtained from commercial sources.
Chromatophores were prepared from photosynthetically grown R. rubrum cells (S-1) (Ormerod et
al., 1961) by grinding with sand (Baltscheffsky, 1960).
Bacteriochlorophyll was determined by the method
of Clayton (1963). ADP-glucose pyrophosphorylase
was partially purified by a modification of the method
of Furlong & Preiss (1969) and assayed as reported
previously (Guillory & Fisher, 1971).
Evaluation of PP1 synthesis
A trapping system, illustrated by eqns. (1) and (2),
was used for the rapid conversion of PPi into a stable
product not susceptible to hydrolysis by the chromatophore pyrophosphatase.
*Abbreviation: ATPase, adenosine triphosphatase.
The glucose 6-phosphate formed by the action of
ADP-glucose pyrophosphorylase and hexokinase was
measured fluorimetrically by the reduction of NADP+
during the oxidation of glucose 6-phosphate in the
presence of glucose 6-phosphate dehydrogenase
(eqn. 3). The formation of glucose 1-phosphate was
determined similarly on the same sample by coupling
with phosphoglucomutase and glucose 6-phosphate
dehydrogenase (eqns. 3 and 4).
Photopyrophosphorylation was carried out at 30°C
with illumination (7.5 x 10-3J S-. cm-2) supplied by
four 100W tungsten lamps. The reaction mixture
contained, in a volume of 1 .5 ml: 70mM-glycylglycine
buffer, pH7.8, l9mM-pyruvate [to activate the ADPglucose pyrophosphorylase (Furlong & Preiss, 1969)],
15mM-KH232P04 (3 x 106c.p.m./,umol), 5.7mMMgCI2, 0.4mg of hexokinase, 50mM-glucose, 70mMsodium ascorbate, 60,ug of oligomycin [to inhibit
ATP synthesis (Baltscheffsky & Baltscheffsky, 1960)],
0.7mM-ADP-glucose, 0.5 unit of ADP-glucose pyrophosphorylase and chromatophores containing 50,g
of bacteriochlorophyll. The reaction was terminated
after 10min by a 5min immersion of the incubation
tube in a boiling-water bath. The sample was cooled
in an ice bath and centrifuged to remove precipitated
membranes and proteins. Portions (0.005-0.1 ml) of
the supernatant solution were taken immediately for
fluorimetric analysis of glucose 6-phosphate and
glucose 1-phosphate (Guillory & Fisher, 1971). The
synthesis of PP1 in the presence of the ADP-glucose
pyrophosphorylase-hexokinase couple results in an
equivalent formation of glucose 6-phosphate and
glucose 1-phosphate. An increment in glucose 6phosphate in excess of glucose 1-phosphate was
taken to represent ATP synthesis. In some experiments glucose 6-phosphate and glucose 1-phosphate
in the reaction mixture was determined by the isobutanol extraction procedure for esterification of Pi
described under 'Evaluation of ATP synthesis'.
Evaluation of ATP synthesis
Photophosphorylation of ADP to ATP in the
absence of the ADP-glucose pyrophosphorylasehexokinase-trapping system was carried out at 30°C
with illumination of 7.5 x 10-3 J cm-2 * s-. The reaction mixture contained, in a total volume of 3 ml:
3OmM-glycylglycine buffer, pH7.8, 3.3mM-MgCl2,
ADP-glucose
PP1 + ADP-glucose
ATP + glucose
pyrophosphorylase
PP
Hexokinase
Glucose 6-phosphate
ATP + glucose 1-phosphate
ADP + glucose 6-phosphate
dehydrogenae gluconolactone 6-phosphate + NADPH + H+
Glucose 6-phosphate + NADP+
Phosphoglucomutase
Glucose 1-phosphate
- > glucose 6-phosphate
(1)
(2)
(3)
(4)
1972
SYNTHESIS OF INORGANIC PYROPHOSPHATE
3mM-ADP, 50mM-glucose, 0.4mg of hexokinase,
16mM-ascorbic acid, 15mM-KH232P04 (3 x
106 c.p.m.//xmol) and chromatophores containing up
to 50,tg of bacteriochlorophyll. The reaction was
terminated after Smin by a 5min immersion of the
incubation tube in a boiling-water bath. The heated
tubes were cooled in an ice bath for 15 min and
centrifuged. A sample (0.1 ml) of the deproteinized
supernatant solution was added to 5ml of 1 %
ammonium molybdate containing 6mM-HClO4 and
the esterification of 32p1 was evaluated by the isobutanol extraction procedure of Lindberg & Ernster
(1956).
Preparation of R. rubrum chromatophores resolved
with respect to ATPase activity
R. rubrum particles resolved with respect to
ATPase activity were prepared by adding 3ml of
chromatophores (about 300,ug of bacteriochlorophyll/ml) suspended in 0.1 M-glycylglycine-10 % (w/v)
sucrose buffer, pH8, to 50ml of solution containing
0.25M-glycylglycine-2M-LiCl, pH 8. After continuous
stirring at 4°C for 10min, the suspension was centrifuged for 30min at 150000g. The sedimented chromatophores were resuspended in 0.1 M-glycylglycine10% sucrose buffer, pH8, to a final concentration
of 300,g of bacteriochlorophyll/ml.
Assay of ATPase and pyrophosphatase activities
ATPase was assayed for 10min at 30°C in a reaction medium (total volume 1 ml) containing: 33 mMglycylglycine buffer, pH8, 96mM-sucrose, 2mMATP, 2mM-MgCl2, 3mM-phosphoenolpyruvate,
0.03 mg of pyruvate kinase and chromatophores containing 30,ug of bacteriochlorophyll. Pyrophosphatase activity was assayed for 5 min at 30°C in reaction
medium, containing in 1 ml: 42mM-glycylglycine
buffer, pH8, 123mM-sucrose, 5mM-MgCl2, 5mMPP1 and chromatophores (30,ug of bacteriochlorophyll). The reaction was terminated by the addition
of 0.1 ml of 70% (v/v) HC104. After centrifugation
samples of the deproteinized supematant solutions
were taken for phosphate analysis (Lohman &
Jendrassik, 1928).
473
Results
Light-dependent PPL synthesis by R. rubrum chromatophores
Fig. 1 illustrates the rate of light-dependent PP1
synthesis catalysed by R. rubrum chromatophores
when esterification of Pi is carried out in the presence
of the ADP-glucose pyrophosphate-trapping system
and in the absence of added ADP. In contrast to the
non-linear rates reported in the literature
(Baltscheffsky & von Stedingk, 1966), linearity of
PP, synthesis is maintained for at least 15min under
the described experimental conditions. Essentially no
PP1 synthesis was detected in the dark. Optimum
rates of PP1 formation were attained by adjusting the
concentration of the components of the trapping
system. Table 1 (Expt. A) shows that the absolute
magnitude of observed Pi esterification can be increased over 10-fold by increasing the concentration
of ADP-glucosepyrophosphorylase. In this particular
experiment, ADP-glucose pyrophosphorylase in excess of 0.4 unit/ml slightly inhibited PP1 synthesis. A
0.20
0.15i
0
1:1.
I)
I)
U,
5
10
15
Time (min)
Assay of energy-linked transhydrogenation
The energy-linked reduction of NADP+ by
NADH was assayed by the method of Keister & Yike
(1967a) by using the reconstituted system described
earlier (Fisher & Guillory, 1969c, 1971a,b). In all
cases saturating concentrations of transhydrogenase
factor were added to the reaction medium.
Vol. 129
Fig. 1. Time-course of PP, synthesis by R. rubrum
chromatophores
The rate of PP1 formation was assayed fluorimetrically as described under 'Evaluation of PP1 synthesis'.
Chromatophores containing 30,ug of bacteriochlorophyll were used. PP, synthesis: *, in the light; o,
in the dark.
474
Table 1. Influence of ADP-glucose and ADP-glucose
pyrophosphorylase on light-dependent PP, synthesis in
R. rubrum
The reaction medium was the same as for Fig. 1
except that chromatophores containing 74,g of
bacteriochlorophyll were added. Incubation time was
for 5min at 30°C in the light and PP1 synthesis was
evaluated by using the isobutanol extraction procedure as described in the Materials and Methods
section. In Expt. A, ADP-glucose pyrophosphorylase
(specific activity 1.36) was added as indicated. In
Expt. B, 1.0 unit of ADP-glucose pyrophosphorylase
was added. PP1 synthesis is recorded as ,umol of
32P1 esterified/h per mg of bacteriochlorophyll. A
unit of ADP-glucose pyrophosphorylase activity is
defined as that quantity of enzyme bringing about
the formation of lumol of NADPH/min in the
standard assay and the specific activity as ,umol of
NADPH formed/min per mg of protein (Guillory &
Fisher, 1971).
ADP-glucose pyrophosphorylase (units) added PP, synthesis
0
0.94
Expt. A
0.968
7.70
0.136
9.86
11.18
0.272
0.408
9.86
ADP-glucose (mM) added PP1 synthesis
0 (dark control)
0.14
Expt. B
0
0.58
0.26
11.40
0.65
9.80
1.29
8.94
2.58
6.66
3.87
7.52
variation in the ability of ADP-glucose pyrophosphorylase of the same specific activity to stimulate
PPi synthesis maximally was consistently observed;
the reason for this variability is unclear. Each ADPglucose pyrophosphorylase preparation was therefore assayed individually and that quantity giving the
maximal stimulation of measured PPi synthesis was
used. ADP-glucose at 0.25mM saturated the trapping
system (Table 1, Expt. B) and omission of ADPglucose (orADP-glucosepyrophosphorylase) resulted
in only low values of "Pi esterification in the absence
of added adenine nucleotides.
The results of a comparison of PPi synthesis by
means of the fluorimetric determination of the
products of the PP1 trap (Guillory & Fisher, 1971)
with an evaluation of synthesis as measured by 32P1
esterification are described in Table 2. In the fluorimetric analysis, the quantity of PP1 synthesized is
R. J. GUILLORY AND R. R. FISHER
estimated directly from the concentration of glucose
1-phosphate, and ATP synthesis can be represented
by the difference in the quantities of glucose 6-phosphate and glucose 1-phosphate. An excellent correlation of the two methods of PP1 analysis is
shown (Table 2, Expt. A). Sufficient oligomycin must
be added to the reaction mixture to inhibit completely
light-dependent ATP synthesis (Table 2, Expt. B).
In the absence of both oligomycin and added adenine
nucleotides some 90% of the P1 esterification represents glucose 6-phosphate formation resulting
directly from ATP synthesis. Under these latter conditions ATP synthesis is assumed to arise from the
ADP supplied by the phosphorolysis of ADPglucose (eqn. 1) followed by the phosphorylation of
glucose by the ATP (eqn. 2). That this is indeed the
case is indicated by the complete lack of even small
amounts of ATP synthesis on the omission of ADPglucose and ADP-glucose pyrophosphorylase (Table
2, Expt. B).
Light-dependent PP1 synthesis in chromatophores lacking ATPase activity
R. rubrum chromatophores can be prepared that
are totally deficient in ATPase activity (Fisher &
Guillory, 1969a,b). Such membrane particles no
longer catalysed the ATP-dependent transhydrogenase reactions or had an active ATPase activity,
but did show PPi-dependent transhydrogenation and
pyrophosphatase activity. The presence of the two
activities is taken to represent the retention of a
functional light-dependent PPI-synthesis system
(Fisher & Guillory, 1969a,b). In Table 3, a comparison is made of the rates of light-dependent synthesis
of ATP and PP1 as well as of hydrolysis of ATP and
PP1 by chromatophores and chromatophores extracted with 1.9M-LiCl (see the Materials and
Methods section). The LiCl-treated particles catalyse neither the photophosphorylation of ADP nor
the hydrolysis of ATP, but retain the light-requiring
PP, synthesis reaction. Since the LiCl-treated particles
catalyse a pyrophosphatase activity of the same order
of magnitude as that found for preparations not
treated with LiCl it is considered likely that the pyrophosphatase represents an activity of the terminal
phosphorylating enzyme of photopyrophosphorylation.
Effect of Mg2' and pH on PPi synthesis
Mg2+ is required for light-dependent phosphorylation of ADP to ATP (Horio et al., 1963). Fig. 2
shows that there is also a Mg2+ requirement for lightdependent PPi synthesis. Maximal rates of PP1 synthesis were obtained at 3.3 mM-Mg2+. At higher Mg2+
concentrations an inhibition of PPi synthesis was
observed. Horio et al. (1963) have reported that
1972
SYNTHESIS OF INORGANIC PYROPHOSPHATE
475
Table 2. Comparison of the radioisotopic and fluorimetric assay of PP1 synthesis
In Expt. A the reaction mixture was the same as for Fig. 1 except for the addition of 60,ug of oligomycin and
chromatophores containing 25 jig of bacteriochlorophyll. For the radioisotope determination of pyrophosphate
synthesis the isobutanol-extraction procedure for 32P1 of Lindberg & Emster (1956) was used as outlined in the
Materials and Methods section for the estimation of ATP synthesis. In this case adenine nucleotide was omitted
from the incubation medium and pyrophosphate synthesis represents one-half of the total amount of phosphate
esterified. For Expt. B the reaction medium was identical except for the indicated changes.
Organic phosphate ester formed (jumol)
Fluorimetric procedure
Omissions or additions
Expt. A None
-ADP-glucose
-ADP-glucose pyrophosphorylase
Expt. B None
+oligomycin (20,ug)
+oligomycin (40,ug)
+oligomycin (60,ug)
-ADP-glucose and ADP-glucose
pyrophosphorylase
Radioisotopic
procedure
0.29
0.09
0.05
Glucose
6-phosphate
0.22
0.00
0.03
2.80
0.53
0.77
0.22
0.00
Glucose
1-phosphate
0.29
0.00
0.00
0.25
0.34
0.36
0.29
0.02
Table 3. Effect of LiCI extraction on chromatophore light-dependent synthesis of ATP and PP1
ATPase and pyrophosphatase activities were assayed as described in the Materials and Methods section. For the
determination of ATP synthesis and pyrophosphate synthesis the fluorimetric procedure was used as described in
the Materials and Methods section. Chromatophores and LiCl-extracted chromatophores each containing 52,ug
of bacteriochlorophyll were used.
LiCI-extracted particles
Chromatophores
(,mol of Pi formed/h per mg of bacteriochlorophyll)
Assay
0
286
ATPase
228
315
Pyrophosphatase
([Lmol of Pi esterified/h per mg of bacteriochlorophyll)
0
276
ATP synthesis
PP1 synthesis
31.4
18.9
maximal ATP synthesis occurs at 3mM-Mg2+, although with ATP synthesis higher Mg2+ concentrations did not decrease phosphate esterification.
The pH optimum for PPi synthesis was found to be
at pH7.5 (Fig. 3) at saturating concentrations of the
ADP-glucose pyrophosphorylase-trapping system.
This compares with the slightly higher pH optimum
of 8 found for light-dependent ATP synthesis (Horio
et al., 1965). For measurement of the pH optimum for
PP1 synthesis, the phosphate buffer that was used in
place of glycylglycine buffer resulted in a much lower
net synthesis of PPI. In fact, at pH7.5 PP1 synthesis
in glycylglycine was some four times faster than in
phosphate buffer. This effect cannot be ascribed to
an inhibition of ADP-glucose pyrophosphorylase by
Vol. 129
phosphate ion, since the R. rubrum enzyme, in contrast to the ADP-glucose pyrophosphorylase from
other cell types, is relatively insensitive to phosphate
(Preiss et al., 1966). A similar stimulation of the
pyrophosphatase activity and of PP1-dependent
transhydrogenase activity was observed in the presence of glycylglycine or EDTA. Such stimulation
may be related to activation of the terminal phosphotransferase enzyme of the energy-coupling system,
perhaps by chelation of a heavy-metal inhibitor.
Influence of reducing agent on PP1 and A TP synthesis
Horio & Kamen (1962) reported that maximal
light-dependent ATP synthesis occurs only when the
R. J. GUILLORY AND R. R. FISHER
476
18r
60
0
.o
to
E 14
0
I-
0
40
s
:o 10
.0
s
0
-
6
40
30
.z
-
Cd
_mu
4.1
0
6
8
pH
Fig. 3. pH-dependency ofPP, synthesis in LiCI-treated
20
v ,
l
o
t
chromatophores
lo
Is
20
[MgCI2] (mM)
Fig. 2. Influence of Mg2+ on PPL synthesis in LiCItreated chromatophores
The assay procedure was as indicated for Fig. 1 with
LiCl-treated chromatophores containing 30,ug of
bacteriochlorophyll. [Mg2+] was varied as indicated.
o
7
5
chromatophores are at an oxidation-reduction
potential of about OV. The external reductant is not
oxidized during ATP synthesis; rather it 'poises' the
components of the electron-transport chain in a
manner that facilitates electron transport and energy
coupling. Fig. 4 shows the dependency of lightdependent PP1 synthesis on the concentration of an
exogenous reducing agent (ascorbic acid) added to
the reaction mixture. Maximal PP1 synthesis was
found at 50mM-ascorbate, whereas maximal ATP
synthesis required the addition of 100mM-ascorbate.
In this experiment the maximal rate of PP1 synthesis
represented about 15 % of that of ATP synthesis. As
the ascorbate concentration is increased above 75mM
the synthesis of PP1 declines sharply and at 300mMascorbate it is only 5 % of the rate of ATP synthesis.
Influence of light-intensity on PP1 and ATP synthesis
A comparison of the effect of light-intensity on the
rates of synthesis of PPi and ATP is given in Fig. 5.
Half-maximal rates of PP1 synthesis were obtained at
a light-intensity of 2.5 xl104J S. cmA2. The halfmaximal rate of ATP synthesis was measured at 5.5 x
10-4J- s- cm-2. Although the PP1 synthesis system
was light-saturated at 1 x 10-3 J - S-1. cm-2, the ATPsynthesis system was not saturated at 9.5x10-3J-
PP1 synthesis was measured as indicated under
'Evaluation of PP1 synthesis' except that 16mMpotassium phosphate buffer replaced the glycylglycine buffer. LiCl-treated chromatophores containing 48,g of bacteriochlorophyll were used.
s-1 cm-2. The ratio of the rates of PP1 and ATP
synthesis ranged with different chromatophore
preparations from 0.5 to 1.0 at low light-intensities
and from 0.10 to 0.25 at saturating light-intensities
for ATP synthesis.
Influence of energy-transfer inhibitors and uncouplers
on PPi synthesis
Oligomycin is an established energy-transfer
inhibitor of mitochondrial oxidative phosphorylation
(Lardy et al., 1958; Lee & Ernster, 1966). That it
functions similarly in R. rubrum chromatophores is
indicated by its inhibitory action on light-dependent
ATP synthesis (Baltscheffsky & Baltscheffsky, 1960)
as well as chromatophore ATPase activity, and its
lack of inhibition of other light-dependent energylinked reactions, including PP1 synthesis (Baltscheffsky & von Stedingk, 1966) and nicotinamide nucleotide transhydrogenase (Keister & Yike, 1967b). These
experiments place the site of action of oligomycin on
the ATP-synthesis system after the formation of the
non-phosphorylated high-energy intermediate (or
high-energy 'state'). The results in Table 2 (Expt. B)
confirm the reported slight stimulation of PP1 synthesis in R. rubrum chromatophores by oligomycin
(Baltscheffsky & von Stedingk, 1966). In LiClextracted particles, oligomycin promoted PPi synthesis by some 300% (Fig. 6), up to the extent of
synthesis found for untreated chromatophores.
Oligomycin at low concentrations has a similar
1972
SYNTHESIS OF INORGANIC PYROPHOSPHATE
477
280
240
30
0
.0
to
W
40
0
v~~
N
~
~
~ ~
0
0~~~~~~~~~0
-
1601.
0.02100.
so
40
1.0
10.0
100.0
0~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~t
1000.0
[Ascorbic acid] (.lM)
Fig. 4. Light-dependent synthesis of PP, and ATP as a function of the concentration of ascorbate
PPi and ATP synthesis were assayed as described under 'Evaluation of PP1 synthesis'. In the absence of added
ascorbate, ATP formation (-) was 63.5,umol/h per mg of bacteriochlorophyll in the light and 0.38,umol/h per
mg in the dark. For PP1 formation (o) the values were 6.91 ,umol/h per mg of bacteriochlorophyll in the light and
0.38,tmol/h per mg in the dark.
influence on R. rubrum reconstituted light-dependent
nicotinamide nucleotide transhydrogenation (Fisher
& Guillory, 1969c, 1971a,b). Table 4 shows that
whereas oligomycin maximally stimulated the reaction in chromatophores by only 10-15 %, the rate of
transhydrogenation catalysed by LiCl-extracted
particles is stimulated some 250% by the antibiotic.
This large-scale stimulation represented 80 % of the
maximal rate observed for control chromatophores.
The influence of oligomycin may be related to the
stabilization of a non-phosphorylated high-energy
intermediate (Lee & Ernster, 1966), which participates
as an energy donor for synthesis of PP1 and ATP, as
well as for transhydrogenation and reversed electron
transport. Possibly the intermediate is rendered
unstable on treatment of chromatophores with
LiCl.
The influence of various uncouplers and inhibitors
on PP1 synthesis is reported in Table 5. Dicyclohexylcarbodi-imide resembles oligomycin in its effect on
chromatophore ATP synthesis, transhydrogenation
and NADI reduction (Keister & Yike, 1967a,b).
However, the mechanism of dicyclohexylcarbodiimide action clearly differs from that of oligomycin
in that it does not stimulate PP1 synthesis in LiClVol. 129
extracted particles. The fact that dicyclohexylcarbodiimide half-maximally inhibited the chromatophore
ATPase activity at 50 tM, whereas ten times this
amount did not inhibit the membrane pyrophosphatase activity, is taken to indicate that the primary
site of dicyclohexylcarbodi-imide inhibitory action
is on that sequence of reactions responsible for ATP
synthesis. The ATPase and pyrophosphatase hydrolytic reactions are taken to represent the reversal of
the respective synthetic reactions.
The antibiotic Dio-9 (Guillory, 1964) inhibits lightdependent chromatophore ATP synthesis, ATPase
and the energy-linked transhydrogenase reaction
driven by ATP, PPi or light (Fisher & Guillory,
1967), as well as being an inhibitor of the reduction
of NAD+ by succinate (Gromet-Elhanan, 1969). It
is a potent inhibitor of spinach chloroplast ATPase
(McCarty et al., 1965) and purified baker's yeast
mitochondrial ATPase (Schatz et al., 1967). Consistent with its inhibitory effect on the PPi-dependent
transhydrogenase (Fisher & Guillory, 1967), Dio-9
has been found to inhibit the light-dependent synthesis of PPi (Table 5). Chromatophore pyrophosphatase activity is inhibited by the antibiotic in a
parallel fashion to its inhibition of PP1 synthesis.
R. J. GUILLORY AND R. R. FISHER
478
0
40
-24 0
4)
*E-a
10
0
0
5
10
15
20
25
30
Oligomycin (,ug)
Fig. 6. Influence of oligomycin on PP1 synthesis in
LiCl-treated chromatophores
0
1
3
2
9.5
10-11 x Light-intensity (J.sS
ScCM2)
Fig. 5. Influence of light-intensity
on
The reaction mixture and assay conditions were the
same as for Fig. 1 except for the addition of LiClextracted chromatophores in place of untreated
chromatophores (48,ug of bacteriochlorophyll) and
20mM-MgCl2. Oligomycin was added as indicated.
the synthesis
of PP1 and ATP
(a) The rates of formation of PP1 and ATP were
assayed as described under 'Evaluation of PPi synthesis', except that the light-intensity was varied as
indicated. The maximum rate of PP1 synthesis (o)
was 40,umol of Pi esterified/h per mg of bacteriochlorophyll and that for ATP synthesis (e) was
250,umol of Pi esterified/h per mg of bacteriochlorophyll. (b) A comparison of the rate of PP1 synthesis to
the rate of ATP synthesis (vPP1/vATP) as a function
of light-intensity, calculated from the data of (a).
Arsenate was found to be an effective inhibitor of
PPi synthesis, with 50 % inhibition occurring at 5mM.
Light-dependent ATP synthesis in chromatophores
has been shown to be inhibited by the uncoupling
agents carbonyl cyanide m-chlorophenylhydrazone
(Geller & Lipmann, 1960), 2,4-dinitrophenol (Bose,
1963) and gramicidin (Baltscheffsky & Baltscheffsky,
1960). The results in Table 5 show that carbonyl
cyanide m-chlorophenylhydrazone inhibits the lightdependent PPi synthesis to an extent similar to that
reported for ATP synthesis. The ionophorous uncoupler valinomycin has been shown partially to
inhibit ATP synthesis (Jackson et al., 1968), but to
have no effect on PP1 synthesis (Baltscheffsky &
von Stedingk, 1966) in R. rubrum chromatophores.
The antibiotic nigericin, although having little in-
Table 4. Influence ofoligomycin on the light-dependent
transhydrogenase reaction
The reconstituted light-dependent transhydrogenation was assayed as described in the Materials and
Methods section. Transhydrogenation is recorded as
mol of NADP+ reduced/h per mg of bacteriochlorophyll.
Wt. of oligomycin Transhydrogenation
System
(,.g)
0
35.4
LiCl particles
44.5
LiCl particles
0.5
1.0
61.9
LiCl particles
73.4
2.0
LiCl particles
10.0
80.0
LiCl particles
0
89.4
Chromatophores
10.0
97.6
Chromatophores
hibitory effect on ATP synthesis itself, synergistically
increases the uncoupling produced by valinomycin
(Jackson et al., 1968). Contrary to a previous report
(Baltscheffsky & von Stedingk, 1966), we find that
valinomycin inhibits PP1 synthesis by up to 50 % and
that nigericin potentiates this inhibition in a manner
similar to its effect on the inhibition ofATP synthesis.
1972
SYNTHESIS OF INORGANIC PYROPHOSPHATE
479
Table 5. Effect of various uncouplers and inhibitors on PP, synthesis in LiCi-extracted particles
Pyrophosphate synthesis was assayed by using the assay conditions and the fluorimetric procedure as described
in the Materials and Methods section. Control pyrophosphate synthesis was 20,umol of P1 esterified/h per mg of
bacteriochlorophyll. Additionally, 14mM-KCl was present in the reaction medium in the valinomycin and
nigericin experiments.
PP1 synthesis
Concn.
(% of control)
Addition
70tM
90
Dicyclohexylcarbodi-imide
85
21O,M
Dicyclohexylcarbodi-imide
65
44,ug/ml
Dio-9
27
Dio-9
170/.Lg/ml
23
7mM
Arsenate
17
14mM
Arsenate
83
Carbonyl cyanide m-chlorophenylhydrazone 0.35M
32
Carbonyl cyanide m-chlorophenylhydrazone 2.8,UM
51
5 ,uM
Valinomycin
5HM
105
Nigericin
5
Valinomycin
5ItM
1 PM
1
Valinomycin+nigericin
Discussion
A number of experimental findings indicate that
the multienzyme systems responsible for the coupling
of electron transport to the synthesis of PPi and of
ATP in R. rubrum chromatophores must be intimately
interrelated: (a) simultaneous synthesis of ATP lowers
the rate of PPi synthesis by as much as 50% (Horio
et al., 1968), perhaps by competition for a common
high-energy intermediate; (b) PPi as well as ATP
provides energy for the formation of a high-energy
intermediate functional in transhydrogenation
(Keister & Yike, 1967a) and reversal of electron
transport (Keister & Yike, 1967b; Baltscheffsky,
1968); (c) PPi hydrolysis can provide the energy for
the synthesis of ATP in the dark (Keister & Minton,
1971).
The extraction of chromatophores with LiCl results
in depletion of membrane-bound ATPase activity and
a corresponding loss of light-dependent ATP synthesis (Table 3) and ATP-dependent transhydrogenase (Fisher & Guillory, 1969a,b) activities. This
treatment, on the other hand, does not damage the
PP,-synthesis system, since the LiCl-treated chromatophores synthesize PP1 at high rates in the light,
possess pyrophosphatase activity equal to or greater
than that of untreated chromatophores (Table 3) and
utilize PPi as an energy donor for transhydrogenation.
A soluble mitochondrial ATPase enzyme first
isolated and described in 1960 is able to recouple
ATP synthesis to oxidation in ATPase-deficient submitochondrial particles (Penefsky et al., 1960).
Although treatment of chromatophores with LiCl
depletes the membrane of ATPase activity, no measurable ATPase activity appears to be solubilized by
Vol. 129
this procedure. A soluble ATPase has, on the other
hand, been isolated from R. rubrum chromatophores,
which when added to a suspension of LiCl-treated
particles reconstitutes membrane-bound ATPase
activity, an inhibitor-sensitive 32PI-ATP exchange
reaction and light-dependent ATP synthesis (Konings
& Guillory, 1971). A similar ATPase enzyme, which
acts as a coupling factor, has been isolated from
Rhodopseudomonas capsulata (Buccanini-Melandri
et a., 1970). Such reconstitution experiments indicate
that treatment of R. rubrum membranes with LiCl
brings about an actual release of the ATPase enzyme
from the membrane, rather than an inactivation of the
enzyme on the membrane. These results support the
concept that two different terminal enzymes, an
ATPase and a pyrophosphatase, participate in the
terminal phosphate esterification reaction during the
synthesis of ATP and PP1 respectively.
Baltscheffsky (1968) reported that the chromatophore b-type cytochrome is reduced by a c-type cytochrome during reversed electron transport promoted
by ATP or PPi, indicating that the primary coupling
site on the electron-transport chain is the same for
both systems. That the two systems may share a
common non-phosphorylated high-energy 'state' is
indicated by the work of Horio et al. (1968), who
observed that the addition of ADP inhibits lightdependent PP1 synthesis by as much as 50 %. Keister
& Minton (1971) reported that PPi could supply the
energy for ATP synthesis in the dark. However, 32p
was not directly transferred to the synthesized ATP,
suggesting that a common phosphorylated intermediate is not formed.
Lipmann (1965) has suggested that PP1 might have
480
been an evolutionary precursor of ATP as the primary
biochemical energy donor, and Baltscheffsky (1967)
noted that the R. rubrum system for coupling PP1
synthesis to electron transport may have evolved
before the ATP-synthetic system. The finding that
these two energy-coupling systems differ apparently
only in their terminal enzymes indicates that such an
evolutionary process may have involved the addition
of a single enzyme, an ATPase, to the bacterial
membrane.
Maximal rates of PPi and ATP synthesis were
found to occur at similar Mg2+ concentrations and
pH values (Figs. 2 and 3). However, significant differences in required reductant concentration (Fig. 4)
for PP1 and ATP synthesis were observed. Intracellular changes in oxidation-reduction potential
during different metabolic states may act as a control
mechanism for diverting electron-transport-chain
energy into phosphate-bond energy in the form of
ATP or PPI. At saturating light-intensity the rate of
PP1 synthesis never exceeded 25 % of the rate of ATP
synthesis. At low light-intensities, conditions under
which the organism may normally be found in
Nature, the rate of PP1 synthesis approached that of
ATP synthesis (Fig. 5). This higher relative proportion
of PP1 synthesis suggests that under certain conditions
the synthesis of PP1 may be as important a pathway
for the energy metabolism of the cell as that of
ATP.
ATP-dependent reactions that release PPi as a
product of substrate activation are well known for a
number of biosynthetic pathways (Kornberg, 1962).
Although many of these reactions exhibit equilibrium
constants close to unity, the hydrolysis of PP1 by
cellular pyrophosphatase has been postulated effectively to direct the overall reaction to completion
(Kornberg, 1962). On the other hand, the establishment of a high intracellular concentration of PP1
could inhibit the same biosynthetic pathways. The
PP1 formed in the process of such activation reactions
could be used for the maintenance of a pool of highenergy intermediates of the membrane PP,-ATP
synthesis systems. This pool of high-energy intermediates could then be utilized as an energy donor
for active ion transport, transhydrogenation, NAD+
reduction or ATP synthesis. The synthesis and
utilization of PPi may thus represent a homeostatic
mechanism, particularly at low light-intensities where
insufficient light-energy is absorbed to drive ATP
synthesis and other energy-requiring reactions at their
optimum rates.
This work was supported in part by Grants (to R. J. G.)
from the United States Public Health Service (AM 10758),
from the National Science Foundation (GB-30566) and
from the Southern Tier Heart Association of New York.
Some aspects of this work we.rc carried out in collaboration
with Miss Louise Y. Lowe at Cornell University (Ithaca,
R. J. GUILLORY AND R. R. FISHER
N.Y., U.S.A.). R. J. G. is an Established Investigator of
the American Heart Association, Inc. We express our
appreciation to Mrs. P. Bilson and Mrs. Kira Fisher for
their excellent technical assistance during this investigation.
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