Plant Physiol. (1967) 42, 697-705
On the Localization of Organic Acids in Acid-Induced ATP Synthesis1 2, 3
Ernest G. Uribe4, 5and Andre T. Jagendorf4
McCollum Pratt Institute and Biology Department, The Johns Hopkins University,
Baltimore, Maryland 21218
Received January 3, 1967.
Suntinary. This study of the penetration and localization in isolated chloroplasts
of some selected organic acids under inducing and non-inducing conditions has shown
that there are 4 distinct relationsh-ips of acid penetration 'to ATP synthesis. Succinic
acid which is effective as an inducer penetrates quite rapidly at pH 4.0 with the time
course coinciding with that of acid-poise as determined by ATP synthesis. As the
pH of stage I is raised (acid more dissociated) the penetratio-n is slower, and the
internal concentration at equilibrium is less. At pH 6.5 where succinic acid is fully
dissociated there is little or no penetration of the dianion. A portion of the succinic
acid (presumably the dianion) is retained in the chloroplasts on pH transition. This
internal acid can be removed by placing plastids back in solution whose pH is less
than 5.
A relatively ineffective dicarboxylic acid. e.g. malonic, penetrates quite tslowly at
pH 4.0. The acid-poise is maximized and declines (possibly due to acid denaturation
of phosphorylating enzymes) much before the internal malonate is maximal. This
dicarboxylic acid also shows little penetration as the dianion and some of it is
effectively retained on pH transition from 4.0 to 8.4.
Acetic, an ineffective acid, penetrates quite well both as the acid and the anion
and is not retained as the anion on transition from ;pH 4.0 to 8.4.
Glutamic acid which produces ATP yields comparable to those obtained with
HCI was found to penetrate very slowly and did not reveal a measturable amount of
retained acid on transition from pH 4.0 to 8.4.
Hind and Jagendorf reported in 1965 that chloroplasts can synthesize a small amouint of ATP in the
dark if taken f)rom an acidic to a basic pH (4).
This phenomenon of acid-bath phosphorylation has
been further sttudied, and conditionis were found
for optimal yields of ATP (5). Prominent among
those conditions is the incltusion of the proper
buffer in the initial acid stage: gltutamic acid at
3 mm provides Ino higher yield than HCl alone at
pH 4, btutt adding stuccinate at 3 m-m increases the
yields 5-fol-d. The accompany inig paper explores
the strtucttural featuires requliredl in ani acid to effect
maximal yields (11).
1 Contribution nulmber 502 from the 'McCollum. Pratt
Institute.
Supported in part bv NIH granit GM-03923; and by
a Ketterin-g Research Award.
3A preliminary account of some aspects of this w-ork
has been published in the Brookhaven Symposium in
Biology: Energy Conversion by the Photosynthetic Apparatus; BNL 989 (C-48) 19: 1967.
4 Present address: Division of Biological Science, Cornell University, Ithaca, New York 14850.
5 Post-doctoral felilow of the National Science Foundationl and subsequently of the National Institutes of Healtlt.
2
The mechani,sm by which these acids cause
higher yields remains a matter of concern. It does
not seem too likely that they serve as substrates
for some specific enzyme, becauise of ,the very broad
specificity described in the accompanying paper.
The broad specificity, on the other hand, is still
consistent with a role in the chemiosmotic mechanism for phosphorylation proposed by Mlitchell
(7, 8) or some variant of it. In this hypothesis
the direct force driving ATP formation is a gradient in electrochemical activ-ity of hydrogen ions
across the grana disc membranes, specificially, more
protons inside than outside. While this gradient
may be established by light induiced electron transport, in our acid-bath phosphorylation it wotuld be
created as a result of the artificial transition from
acid to base. In the acid stage protons presumabiy
enter the interior regions of the grana; when
placed in an alkaline environimeinft the required pH
gradient and/or membranie potential is automatically
assutred.
The yield of ATP, according to this concept,
would depend both on the pH gradient between
inside and outside, and on the actual number of
internal protons. The number of free protons that
697
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6,98
98LANT P'HYSIOLOGY
Coiild enter in the acidl stage wo;ild certainly be
limited by their concei-ntrationi in the external
meditim; at pH 4.0, this w\ouldl be 0.1 mnI. The
number wotuld of couirse be higher if bouind carboxyl grouips having the right pKa were present
on the inside. If an uindissociate(d organiic acicl was
present at pH 4.0, it might easily enter together
with its protons; these w\ouild serve as a reservoir
w;hen the external mediutm w-as made alkaline.
For instance, if 10 mm sutcciinate xwas added at pH
4.0, it could provide a soulrce of bound, presuimably
penetrating and later dissociable protoins at a maximal concentration of 16 mMi. (This concentration
is calculatedl from the know n pKa valtues for each
of the carboxyl grouips of suiccinate; 1 of them is
completely uindissociated, the second is 60 % undissociatecl at pH 4.0. The calculation fuirther
asstumes that both the total su1ccinate concentration
and the percent d(issociationi become equlal inside
and otutside the grana disc membranes). This is
160 times the concentration of hydrogen ions (10-4)
available in the external medliuim at this pH.
If this hypothesis is correct, the effectiveness of
the acids shouild be a matter of having the right
set of pKa's and of being mo(lerately permeable
in the grana (lisc membranes. These requirements
might easily lead to the observed broad struetuiral
specificity. If the acid couldl not enter at all at
pH 4, it would noit contribulte to the trans-membrane
proton gradient. On the other handl if the uindissociated acid moved too rapi(dly through the
membranes, it might leave so rapiclly as the chloroplasts were brought to pH 8.4 that the protons
wouild not contribute 'to maintainiing the pH gradient.
A precliction from this model is that effective
organic acidIs shotldel be fouind to move inisi(le the
broken chloroplast membranes at pH 4; and some
of the dissociated anion might be left insidle at
pH 8.4. Non-e,ffective acidls might either not penetrate, or else might move throuigh the membranes
too rapidly. The present paper presents data relevant to these quiestions of locializatioin of the organic
aci(ls tinder induicing anld( non-ind(uticing conldlitioins
for acid-base ATP synthesis.
After iincuibatioin with an organic acidI at pHr
4.0 the chloroplasts shouldl probably not be described as being at a high energy level. They have
rather been poisedl at a level of acidity which is
energetic in relation to the alkaline pH imposed
later. We wotild accordingly like to adopt the term
"acid-poised" as an abbreviation for chloroplasts at
the end( of stage I of this (lark, 2-stage phosph,orylation procedulre.
Materials and Methods
Chloroplasts were prepared from market spinach
as described previously (5), broken in 10 mai NaCI,
then centrifuged and resuispended in 10 mat NaCl at
the same chlorophyll concentratioin as that (luring
the original breaking. These were centrifuigedl
again; resuispenidedI in 10 mMi NaCl at a chlorophylI
concentration of 1 to 1.5 mg/ml; aind finally centrifulged for 25 secondls at 0° and 4700 X g uisinlg
t,he high speed attachment of an International PR-2
centriftuge. The stupernatant fraction containing
the light chloroplast fragments was (Irawin off anid
diluted to 0.5 mg chlorophyll per ml; the pellet
Heavy fraction w-as discarded. Insertioin of the
second wash with 10 mai NaCl pro(liices a higher
yieldl of Light chloroplast fragments which were
previouislyinotedl to be the more active part of the
preparationi (6). Chlor,ophyll was (leterminle(l by
the method of Arnon (1).
Acid-bath phosphorylation reactionIs wN-ere carried ouit rouitinely as described in the prexviouis
commuinicationi (11). To assess dlecay of the acidpoisecI state onl resuispension w ithout s;uccinate,
experiments were performed following the protocol
showni in figuire 1. After a rapid centrifugation,
acid-poised chloroplasts wvere resuispended by uise
of a Vortex mixer either in (a) 1.8 ml containinlg
the usual alkaline, phosphorylation stage components, or (b) 0.9 ml of ia fresh aci(d mix at pH
4.0. In the secondl case, after a variable period of
time the alkaline phosphorylation stage components
in 0.9 ml were adlded to the re-poised chloroplasts
still being agitate(d in the Vortex mixer. In both
cases 0.2 ml of 20 % trichloroacetic acidl w,as added
after 15 seconds. ATP wvas assaye(d 1 the method
of Avron (2).
Experiments to determine the interiual concentration of organic aci(ds in acid-poised chloroplasts
(stage I) were carried ouit according to the protocol
showrn in figure 2. In this method chloroplasts
were incubated with '4C-labeled orgainic acid and
3H-labele(d intilil, then centrifuged to yield Pellet I
and Suipernataint I. The total time of exposure to
radioactive componll(ds, incluiding the time for
centriftigation, was 6.5 minutes. Dtuplicate tuibes
without tracers were handlle(d in parallel to permit
an estimate of the total liquii(d voluime of the Pellet
I fraction, by the difference betweeni its w et and
dry weights. Pellet T from this first ceiitriflugation
Chloroplast fragments
in
10 aM succia te pH 4.0 or 4.8
4700 Xg
25 sec.
Pellet I
either
Supernatant I
(aResuspend
or
in
ADP, '2PLMg2+
pH 8.4, 15 sec.,
add 20% TCA
(b)
Resuspend
in
fresh acid mix
pH 4.0, (x) sec.
Add
ADP, 32Pi, Mg2+
pH 8.4,
15 sec.,
add 20% TCA
FIG. 1. FlowN- diagram: me-thod of determination of
effect of centrifuigatioin on azid-poise of chloroplasts.
TCA = triclhloroacetic acid.
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URIBE AND JAGENDORF-LOCALIZATION OF ACIDS IN CHLOROPLASTS
Chloroplast fragments; variable pH
'4l
3
_C-organic a-lid, H-inulin
19,QOOXg, 3.5 min.
.,upernatant I
(di.card)
Pellet" I
l
reigh;
of
r<u.
,
-n
L.", ral ;, ,2ST Tricine ?H 0.4
.L9 ,,CC XE-, 3.
nminm.
-uen- - nr. v '
TCA; centiiXu-e
r-u
Suuernatant III
f or"C-acid
-inulin
Pellet III (--.
A.--s a
-tnd
FIG. 2. Flow diagram: method of determination of
internal acid under inducing and non-inducing conditions;
and also for determination of time course for penetration
of organic acids. See methods for further explanation.
TCA = trichloroacetic acid.
resuspended in 55 mm Tricine at pH 8.4 and
re-centrifuged, yielding Pellet II and Supernatant
II. Pellet II was resuspended in 2 % trichloroacetic acid and re-centrifuged so that any internal
acid would be released into Supernatant III. Radioactivity in Supernatants II and III was assayed by
placing 0.2 ml aliquots into 10 ml of Bray's solution
(3) and counting with a Packard Tricarb liquid
scintillation counter.
In this experiment it is presuimed that the chloroplast Pellet I, after the first centrifugation, contains both external surface water and internal
water. The inulin content (counted in the wash
Supernatant II) is taken as a measure of the volume
of external water. From this volume the amount
of succinate external to the chl-oroplasts in Pellet I
can be calculated (assuming that both succinate
and inulin are at the same concentration in the
surface film as in the original solution).
The internal organic acid of Pellet I will be in
part retained, and in part lost to the medium when
these chloroplasts are resuspended at pH 8.4. Centrifuging the resuspended Pellet I thuis separates
into Supernatant II both the organic acid which
was external to begin with in Pellet I, and that
which leaks out at pH 8.4. By subtracting the
calculated value for external organic acid of Pellet
I (-see above) from the to,tal in Supernatants II
plus III, we are left with an estimate of the internal
was
acid present in Pellet I before the transition to
pH 8.4. Pellet II, and its acid extract (i.e., Supernatant III) reveals directly the organic acid which
is retained even after transition to a basic pH.
The capacity of the acid-poised chloroplasts to
accomplish ATP formation was determined by the
routine method on a separate aliquot (not shown in
the flow-sheet of fig 2) using a 20 to 30 second
incubation in stage I (11). Control experiments
699
showed that succinate entry is complete in 20 to 30
seconds (fig 4) and then shows no further decline
up to 6.5 minutes in the acid stage. This is so even
thotugh the capacity to form ATP may decline
considerably between 30 seconds and 6.5 minutes
(see figs 4 and 5). Since the amount of internal
succinate is the same at 0.5 and 6.5 minutes, it is
valid to compare this later value to that of the
acid-poise (i.e. ability to make ATP) measuired
after 20 seconds of acid incubation.
The time coulrse for penetration and retentioni
of organic acids under varying conditions was determined by a modification of the procedure of
figure 2. In experiments on penetration rate chloroplasts were incubated with either the labeled
organic acid only, or labeled organic acid and
labeled inullin, for varying lengths of time. In both
single and double label experiments they were
brought to pH 8.4 in the usual way. When the
single label method was used, the chloroplasts at
pH 8.4 were carried through the protocol of figure
2, through the first 2 centrifugations. Rather than
add trichloroacetic acid and centrifuge a thi,rd time,
however, Pellet II w,as simply resuispended in 0.5
ml of tricine buffer (pH 8.4), plated oni planchets,
dried and coulnted on a Nuclear Chicago gas flow,
thin window detector. \WThen the double lable
methodclas used, the chloroplasts at pH 8.4 were
carried through the protocol of figture 2 to vield
Pellet I. It was resuspended in 1.0 ml of 5 mMN
glultamate (pH 4.0) and centrifuged as shown to
yield Pellet II and Supernatant II. This resuspension effected the loss of internal acid which
had been retaine(d on transition to pH 8.4. Supernatant II was assayed for inul-in and acid by the
liquid scintillatioin technique. The external acid of
Pellet I was calcuilated from the inulin content:
and total organic acid of Pellet I from the amount
in Supernatant II: internal organic acid retained
at pH 8.4 was obtained by difference between total
organic acid and that in the external volume of
Pellet I. Note however, that in this case the
calcuilation applies only to the internal organic acid
surviving after transitioni to pH 8.4. In these experiments a parallel time coulrse for ATP synthesis
as a function of time in the acid stage was carried
out simultaneously uising the routine method.
Suc-cinic acid-2,3-14C, malonic acid-2-1"C an(l
acetic acid-2-14C as the sodiuim salts; DL-glutamic
acid-3,4-14C andl methoxy-3H-inulin were obtained
from the New- England Nulclear Corporation, Boston, Massachusetts.
Results
The resuilts reported in table I show that chloroplast fragments cain be inctubated in succinic acid,
centrifuged, and resuspended either in their own
supernatant or in fresh succinic acid, and retain
their acid-poise condition unchanged. They may
also be resuspended directly in ADP and phosphate
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700
PLANT PHYSIOLOGY
T-ble I. A TP Synthesis by Resuspended Acid-Poised
Chloroplast Pellets
Reactions were run according to the protocol shown
in figure 1. In this experiment Pellet I was resuspended
in 0.9 ml of the acid resuspension mixes shown in the
table below-. Chloroplasts w-ere incubated for a total of
2.5 mmi in stage I prior to their resuspension. The
resuspended chloroplasts were allow-ed to remain for 5
sec in the second acid mix before addinig the phosphorylationi components in 0.9 ml.
ATP
Original
Conc
acid
mm
Stuccinic
10
Succiniic
10
Stuccinic
10
Glultamic
3
Suiccinic* 10
Resuspension
mix
Supernatant I
Gluitamic
Fresh succinic
Freslh gluitamic
Phosphorx lation
Conc
mm
mgmoles/mg
chlorophyll
10
112.2
57.1
107.0
21.7
112.2
3
10
3
mix
Gilutamic*
*
3
Phosphorylation
mix
28.6
These pellets resuspended directly in 1.8 ml of the
phosphorylation mix. In all cases the phosphorylation
reaction was allowed to proceed for 15 sec. All of the
reactions contained 0.25 mg chlorophyll.
siduial suiccinic acid, carried along in or on the
chloroplasts. It is interesting that the final acidpoise level observed is that expected from an
original succinate concentration of 1 mm in the
acid stage (fig 3) whether the experiment was
performed at pH 4.0 or 4.8. This value is obtained
by comparing the residual acid-poise wvith the curve
for acid-poise as a function of stuccinic acid concentration at the partictular pH tusedl.
These experiments establish a stroing presumption that the effective organic acid is that which
penetrates the interior of the grana membrane,
the amount being governed by an equilibrium with
the external acid concentration. The slower onset
and decay of the poised state at the higher pH
further implies that the acid moves freely through
the membranes when uindissociated, but slowly or
not at all when ionized.
The next step seemed to be to try to find direct
evidence for penetration of the organic acid, in
Stage I; and to find correlations between the
amount of internal acid andl the degree of acidpoise indicated 'by ability to make ATP oIn a pH
Succinote Conc., mM
3
5
7
10
at pH 8.4, and cause the formation of just as much
ATP as those resuspended in stuccinate. If resuspended in glttamic acid, however, a considerable
amount of the acid-poise is dissipated. These experiments rule out the need for a soltuble stupernatant factor left over from the acid stage, in
order to make ATP. Thev (lo Inot, however, distinguiish between an internal anid an external ftunctioIn for the sutccinic acid. If sticcinic acid exerts
its effect when external to the chloroplast fragment
membranes, a studden change in suiccinate concentration as by dilution should lead to an immediate
drop in the degree of acid-poise. If, on the other
hand, the acid acts only after it enters the chloroplast inner space, uipon diluition we woulld expect
the rate of diffuision of succilcic acid from inside to
ouitside to be limiting for the rate of drop in
demonstra,ble acid-poise.
txperiments were therefore donie according to
the protocol outlined in figuire 1. Figure 3 shows
there is a gradual loss of the poised state when
chloroplasts, previously incubated in succinic acid,
are resuspended in 3 mm gluttamic acid at either
pH 4.0 or pH 4.8. The loss at pH 4.0 is pseudofirst order with an approximate half time of 1
second; the time for one-halif loss at pH 4.8 is 5
seconds. The slower loss of the poisedl state at
pH 4.8 is directly comparable to the slower onset
of the poised state at pH 4.8 seen in previotusly
published experiments (5).
When chloroplasts are resu,spended in glu-tamate
only (fig 3), the ability to make ATP does not
decline to zero, or even down to the amotunt expected from glutamate alone. It seems probable
that the final acid-poise level is governie(d by re-
5
7
Seconds Resuspension
FIG. 3. Time course for decay of acid-poised state
at pH 4.0 and 4.8. Decay reactions were run usinlg the
protocol of figure 1. In this experiment chloroplasts
containing 0.25 mg chlorophyll were incubated in 0.9 ml
of the standard succinate mix at pH 4.0 or 4.8. The
chloroplasts w-ere incubated for 2.5 mim iii stage I inlclusive of centrifuga'tion. Pellet I was resuspended in
0.9 ml of 3 mm glutamic acid containing 27 m,moles
of DCMU for the time show n1 before adding the phosphorylation components in 0.9 ml. Zero time represents
poised chloroplasts resuspenided directly in 1.8 ml of the
phosphorylation mix. The succinate concentratioin curves
were run using the standard procedure outlined in the
methods sectioni of tlle accompanying paper (11).
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URIBE AND JAGENDORF-LOCALIZATION OF ACIDS IN CHLOROPLASTS
transition. The protocol of figure 2 (see Methods
sectionr) was used with variouis acids, and some
restults are shown in taible III. Note that Pellet I
refers to chloroplasts centriftuged out of the acid
stage: Pellet II is that of chloroplasts centrifuged
after a transition to pH 8.4. In other experiments
(not shown here) suc;cinic acid remaining in Pellet
II was found to be tenaciotusly retained over considerable periods of time and even after fturther
washes at pH 8.4. It was lost rapidly and completely if the chloroplasts were brought back to
pH 5 or below. We can infer therefore that the
succinate anion is trapped inside the membranes
because of its charge, not becauise it is metabolically
assimilated.
Table II shows some representative data, of
tritiated inulin and 14C-labeled succinic acid cotunted
in the fractions described in figure 2. It is apparent that suiccinic acid penetrates chloroplast fragments to a greater extent thian inulin does. We
assume that this organic acid has entered some
internal region of the memtbranes and is not simply
in the adheriing ouitside solution.
Turning to a variety of organic acids (table
11H1) we see that they all penetrate chloroplast
fragmenits to a greater extent than inulin does,
with considerably more entering at pH 4 than at
6.5 or 7. There are significant quantitative dif-
701
ferences between effective and ineffective acids,
however, with respect both to the amount penetrating in the acid stage (Pellet I) and that retained after a pH transition (Pellet II). Succinate,
which is a very effective acid, and malonate, moderately effective, penetrate quite well and up to
15 and 32 % of their original amouint is retained.
Acetic acid penetrates to a much greater extent at
the low pH, but ruishes out allmost completely on
transition to pH 8.4. Glutamic acid, also ineffective, enters to a mtuch smaller extent in Pellet I
(none at all, at pH 6.5); and the total amotunt
retained at pH 8.4 is very small, even thoutgh it is
10 % of the original valtue.
The apparent internal concentrations of the acids
at pH 4 are qutite dissimilar, and none of them
seems to come into equiilibrium with the external
concentration of 10 mm. In nulmerouts other experiments with succinate we have fouind that apparent internal concentration varies from experiment to experiment with a range from 2.98 to
5.90. The most usutal values have centered around
4.5 mm, with a 10 mm external concentration.
The present estimates of internal water voluime
and acidic concentration cannot be considered accurate, however, because they depend oIn the presumption that any water in the chloroplast pellet
not accessible to inulin is inside the grana discs
Table II. Comparison of the Inmulin and Succinate Volumte of Peclet I
Data fromi experiment I table III pH 4.0.
Solution
Pellet I
Total
cpm/ml
29,500
342.000
Inulin
Sutcinic acid
,g/ml
222
...
,u.moles/ml
...
10
counts
596
14,000
Volume
0.020
0.041
Table III. Estimate of Intcrnal Acid of Acid-poised Chl/orop/asts
All experiments were run using 0.25 mg chlorophyll in stage I. In experiments 1, 2 and 3 the reaction mix at
pH 4.0, 6.5 or 7.1 (stage I) contained in 0.9 ml: acid, 9.0 ,Amoles (plus carrier free '4C-acid with approximately
250,000 cpm); inulin, 200 Ag containing 1 ,ucurie 3H-inulin; DCMU, 27 mumoles. The total time of exposure to the acid
pH was 6.5 min inclusive of the first centrifugation. Experiment 4 was done with the same protocol as 1, 2 and 3
except that the conceintration of glutamic acid was 5 mM in stage I. All total amounts are shown on a m,mole per
mg chlorophl ll basis.
Expt
pH
4.0
4.0
4.0
4.0
6.5
7.1
6.5
6.5
no.
1
2
3
4
1
2
3
4
Acid
Succinic
Malonic
Acetic
Glutamic
Succinic
Malonic
Acetic
Glutamic
Internal acid
Pellet I
Pellet II
m,umoles per mg
of chlorophvll
1016
156
748
284
2880
48
316
32
48
268
100
0
692
16
8
0
Pellet I
Apparent
acid conc
82
41
7
23
Internal
water
ml
0.34
0.44
0.52
0.38
0
0
0
0
0.18
0.23
0.24
0.22
1.50
0.44
ATP
yield
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Copyright © 1967 American Society of Plant Biologists. All rights reserved.
mm
2.98
1.70
5 50
0.83
2.90
0.04
702
PLANT PHYSIOLOGY
and accessible to organic acids. It seems very
likely though, that an unknown fraction of the
non-inulini water is actually bound to membrane
colloids, and that this water of hydration is also
inaccessible to succinic and other organic acids.
In this case the estimated organic acid internal
concentrations are all 'too low, although the total
amounts per nmg chlorophyll are accuirate.
The amounlt of sticcinate retaine(d in the chloroplasts at pH 8.4 (Pellet 11) has vraried from 156
to 280 m,tmoles per mg chlorophyll; here the uisual
values have been near 160 m,umoles per mg of
chlorophyll. Starting at pH 6.5 the amount of
succinate retainie(d in chloroplasts at pH 8.4 has
varie(l from 0 to 48, w\ith usulal values on the order
of 10 to 12 mMmoles per mg of chlorophyll.
A fuirther importaint correlation woould be between the kinetics of acid pecetratiou, and that of
rise in acid-poise. It N-as not feasible in these
experiments to use the protocol of figures 1 or 2
and measuire the rate of peinetratioui of total organic
aci(Is at pH 4.0, because the rise in acid-poise is to
a large extent complete in 10 seco,ndls, before any
centrifugation is possible. Howeveer, internal acid
which is retained at pH 8.4 is fixed at its final
concentration as soou as the chloroplasts are made
alkaline; hence it was possible to measutre -the
amount of this "pH 8.4
retained acid" as a
function of time at pH 4, in the early seconds after
exposuire to labeled succinate at pH 4. This is
probably the more signiificant data in anly event,
since the ATP yieldl induced by different acids
shows a closer correlation with the amounlt retained at pH 8.4 than with the initial amount
penetratinig (compare the fairly high amounts of
acetate in Pellet I at pH 4, or of any acid in Pellet
I at pH 6.5, with the low ATP xield).
Experiments were conducted with '4C-labeled
acids or '4C-labeled acids andl 3H-inuilin added to
an acid stage of variable length of time. Figure 4
shows the closely corresponding cuirves for buildulp
at pH 4 of succiinate which is Inot lost at pH 8.4,
and for the ability to make ATP. OIn the other
hand chloroplasts puit into stuccinate at pH 6,5
acquire only 17 % that which enters at pH 4
(fig 4). These (lata together with the previously
mentiolned retention of internal succinate at pH
8.4, indicate that the succinate di-anion does not
readily penetrate the grana membrane from either
direction. The figures for succinate retention
shown in the graph are minimal as fuirther experiments showed that a small amouint of internal
succinate is lost on washing the chloroplast pellet
with Tricine at pH 8.4, probably (Iiie to mechanical
breakage. WNe can calcuilate a suiccinate/ATP
ratio of 2.9 from the data of figuire 4. In other
experiments however, internal succinate/ATP ratios
as low as 1.9 (table II and experiment 2, table IV,
ref. 6) were seen. We believe the variability lies
in ATP synthesis, which in some batches (fig 4)
bult Inot others, show\s anl lunstable time couirse. If
-
Stage
I, pH 4.0
Internal Succinate
80
160
140
70
120 o
to
'&60
a
c 50
a
40
80
@ 30
E
60
E 20
E
40
I0
Stage 1, pH 6.5
Li
t
10
0
0
20
A
l~~~~nternal Succinate
40
60
80
Seconds
20
100
120
FIG. 4. Time course for the penietration of succinic
acid and ATP synthesis. The penetration curve was
determined using chloroplasts containing 0.25 mg chloroph-yll. The chloroplasts were incubated in the standard
10 mm succinate stage I reaction mix which contained
carrier free succinic acid -2,3-14C (approximatelv 250,000
cpm) for the time shown. The chloroplasts were then
transferred into 55 mm tricine containing sufficient
NaOH to neutralize the acid and aclhieve a final pH of
8.4. The chloroplast suspensions w,vere carried through
the modified protocol of figure 2 as outlined in the
methods section and the retained acid determined by radioassay. The acid-bath phosphorylationi time course with
succiniate was run with chloroplasts containing 0.25 mg
chlorophyll. These reactions were run according to the
stanidard procedure given in the metlhods section of the
accompanying paper (11).
the time cotirses are carried ouit ait pH 5.2, one
the same type of correspondlence between ATP
synthesis and sticcinate retentioni, althouigh ith a
longer half-life for both.
In these experiments ATP synthesis reached a
maximtim at 15 to 30 seconds andl then decreasedl
with a half-life to 2 to 3 mintites, while sticcinate
retention always reached a plateaui. The decrease
in ability to make ATP is therefore not duie to loss
of the in,ternal acid, but rather comes from the
prolonged time at the acid pH. A'We have fotiind
(F. G. Uribe, tinpulblished experiments) that chloroplast fragments can synthesize (lecreasing amounts
oif ATP oIn repeated acid-base transitionis, if the
aci(l stage is short (i.e., 15 sec). TPhese bits
of evidence suiggest that a progressive inactivation
of the phosphorylating enzymes may occur at the
acid pH's. The extent of this inactivation is likel)
to be one of the important variables affecting ATP
yield in spinach from different srilrces: in other
sees
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URIBE AND JAGENDORF-LOCALIZATION OF ACIDS IN CHLOROPLASTS
140
Stoge1, pH 4
120
-120
0
o
Internal Molonate
°. 100
/
00
E
°°
880 t
]804
r
40
No SHTosto
E
20
~
~
~
~
~
~~~~~~~2
.
0
30
I
60
120
90
SecondS
150
180
FIG. 5. Time course for the penetration of malonic
acid and ATP synthesis. The penetration curve was determined using chloroplasts containing 0.25 mg chlorophyll. The chloroplasts were incubated for the time
shown in the standard 10 mm malonate stage I reaction
mix containing approximately 250,000 cpm carrier free
malonic acid-2-14C and 200 /Ag (1 /Ac) methoxy-3H-inulin.
The pH transition was done as for the experiment of
figure 4. The determination of the acid retained at
pH 8.4 was done by the protocol of figure 2 as modified for a double label experiment (see Methods). The
acid-bath phosphorylation time course u-ith malonaLe wX-as
done as in the experiment of figure 4.
experiments (5) we have noted a half-life as long
as 10 minutes for the decay in the acid stage.
Double label time course experiments were carried out to assess the rate of penetration and retention of relatively ineffective acid's at inducing
and non-inducinig pH's. In the case of malonate
(fig 5) penetration of the acid was rather slow,
and did not reach a maximum even after 7 minuttes
of incubation. The ability to form ATP rises
(together with the 'level of internal malonate) for
the first 20 to 30 seconds, but then drops rapidly
with a half-life of about 150 seconds.
On turning to the relatively ineffective glutamic
acid, the amounts retained at pH 8.4 are so small
as to be unreliable, but at least there was no sign
of continuing increase with time. This correlates
well with the ATP yield, which shows a very slow
rise to low values (5). In the case of acetate no
rise of retainable acid occurs with time and the
ATP yields are even lower than those found with
glutamic or HCl.
Discussion
Several points emerge from the present study.
The first is a practical one, acid-poised fragmented
703
chloroplasts may be centrifuged and resuspended
withotut loss of activity (table I). The second is
that organic acids clearly do penetrate the broken
chloroplasts, to a space not accessible to inulin
(table II). Penetration into this space, and egress
from it, is much faster as the pH is dropped to
4.0 (figs 3, 4) prestumably because the tindissociated
acid is the penetrating species. The third point is
that a fairly good series of correlations can be
found between internal organic acid content and
yield of ATP in acid-base experiments; and the
variation between different organic acids and different pH's can be explained in part on this basis.
Correlations between acid-poise and internal
organic acid content come primarily from time
courses following sudden variation in the external
concentration of succinic acid. Circumstantial evidence, first of all, is found in the pH dependency
of the onset (5) and loss (fig 3) of acid-poise,
following respectivelv addition and withdrawal of
succinate. Both of these are 5 times faster at
pH 4.0 than at 4.8. Secondly there are the direct
measurements of the time-course of rise in acidpoise and of rise in internal succinic acid at pH
4.0 (fig 4), which are in remarkably good agreement. Third is the low internal succinate and
absence of acid-poise when chloroplasts are incubated at pH 6.5 (table III, fig 4). Internal organic
acid here refers primarily to the suiccinic acid which
is retained even after dilution at pH 8.4. Th.s
consists most probably of the dianion species, because little penetration of the fully charged succinate occurs from outside 'to inside (fig 4). As
far as the few data now on hand are concerned,
it appears that an effective molecule is one which
can penetrate into the inner space o,f the broken
chloroplasts at the low pH, and is to some extent
retained as the anion at the higher pH.
The function of organic aCids could a priori
depend on 1 of 3 factors: the specific nature of the
penetrating molecuile, the proton that it brings with
it at the low pH, or the fact that it has a negative
charge after dissociation at 'the high pH. If the
proton is the important factor, then the correlation
noted above depends on the coincidental fact that
the acid after dissociation is a non-penetrating or
poorly penetrating molecule. If presence of the
charged internal anion is the crucial factor, then
the correlation is a direct one between the amouit
of the effective species and the final yield.
If configuration of the carbon skeleton is not
important in itself, it may seem odd that the
specificity for this function is not more broad.
The study of just 3 poorly effective acids (malonate, glu-tamate, acetate) gives some indication of
complexity in secondary characteristics that could
easily be limiting. Glutamic acid appears to be an
extremely slowly penetrating molecule (fig 2; ref
5) which simply never reaches a sufficiently high
internal concentration, in the ordinary dulration of
these experiments. By contrast the very effective
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704
PLANT PHYSIOLOGY
acid (fig 4) penietrates very rapidly,
reaching a maximum at the same time that the
acid-poise reaches an optimum. Shortly thereafter
the abili,ty to imake ATP declines again, presumably
due to denaturation of some sort. It cannot be a
denaturation brought about by the low pH alone,
however, because chloroplast fragments in HCl or
gltitamate dlo not show a similar loss of ability for
a considerable length of time.
Going oIn to malonate, the rate of penetration is
very muich slower than is that for succinate (fig
5). Although acid-poise rises concurrently with
malonate penetration the loss of ability to make
ATP se-ts in long before the maximum amount of
malonate has entered. One wouild expect in chloroplasts not show%ing such a marked decline with
time in the acid stage (5) that malonate would be
a relatively more effective acid.
Acetate is the most intriguing of all. We do
not know the rate of penetratioin of the uncharge(d
acid ait pH 4, becautse essentiallI none is left after
dilution at pH 8.4 (table III). The anion (i.e. at
pH 6.5) appears to be quite permeant in the chloroplast membranes, (table III) a result reported
previously by Packer and colleagues (9). Vast
amotunts of the uncharged acid do enter at pH 4,
and we wNotild guiess that peneltration is rapid as
well as easy. It is quite ineffective as an acidpoise agent; and this could either he becautse egress
of the free acid is much more rapid than coInsumptioni o'f its protons in phosphorylation; or
because ATP formation reqllires the anion to be
suiccilnic
left
behind(
and
this
simply
(loes
not
occtir
with
acetate.
remembered that the role of the
cutrrently postuilated, is to serve
as a reservoir of protons and thereby prolong the
effec,t of a change in pH. The actutal driving or
proton-motive force (8), the height of the reservoir, is set by the difference in proton concentratioIn between the 2 stages. Thuis althotigh a third
as much sticcinate may enter at pH 6.5 as at 4.0
(table III, compare lines 5 and( 1), the pH difference of 2 tinits from the alkaline stage is not great
enouigh to lead to any ATP formation.
The discussion to this poiInt, and the plan of
the experiments, has been based on the premises
that A) effective acids w-oulcl have at least 1
molecular species capable of penetrating the membranes, and B) that penetration ould be to an
internal aqtieotis phase where association and dissociation reactions wotuld be tinhindered. With
the,se assuimptions it would be expected that in the
abseince of energy input, the external and internal
concentrations wouild come to ecqnilibrium by simple
diffuision, depeinding in part on the respective internial andI external pH's. Indeed, asstumiptions of
this sort form the basis of 1 method of juidging
intracellular (12) and intramitochondrial (10) pH's.
Ouir data do not seem to conform to this simple
pictulre. Althotigh the actuial iinterinal coinceintraIt mtist
be
organic acid,
as
tions of acids are still uncertain dcue to lack of
information on the proportion of bound rather than
free internal water, still no acid seems to come to
the same concentration inside as otitside. If we
examine the amount of internal suiccinic acid as a
fuinction of pH (table III and(I nulmerouis uinpublishe(d experiments) it is seen to penetrate readily
and come to an end-point at pH 4. At higher pH's
the penetration is slower, btit a clear eiil-poinit
occuirs at much lower internial amounlits, even
thouigh the external concentrationi is 10 mI11t in all
cases. Thtis alithouigh suc-cinate might have reached
the same concentration inside as outside at pH 4,
it clearly could not have done so at higher pH's as
well. Other data indicatiing lack of equilibration
between 2 aquieouis compartments comes from the
large variation in apparent internial concentration
between 1 organic acid and another, compare for
instance, succinate and acetate in table II, both of
them readily penetrating species at pH 4 ad(I 1)oth
coming to definite buit unlike end-points of iinternial
concentration. There is also the matter of the
large variation between apparent initernal concentrations of sticcinate from one experimeint to the
next, as if the amouint of internal suicciinate -were
not rellated to internal water voluime as we ctirrently measure it.
Some of the above results couild le explainied
if the internal pH of the chloroplasts were fixedl
at 4 or below, and this valuie varied from 1 batch
to the next (althouigh this sitill would no,t explain
the difference between succinate and acetate in 1
experiment). However, it seems to tis extremely
unlikely that the internal aqueotis phase of chloroplast fragments (previously exposed to 10 mar
NaCl at pH 6.5, more or less) can be maintained
uinder these metabolically inert conditions at pH
4.0. An alternativ-e possibility that shoulld be coInsidlered seriouisly is that at least a part of the
organiic acid's might be stored not in a free aquieous
phase, but rather in a lipid phase where dissociation is hindered and the equlilibriuim is betw-een the
uncharged acid inside anid the iunidissociate(d species
in sol1tioii.
Acknowledgment
The autilors acknoNN-ledge withi tha.inks the expert
tecliical assistance of Mrs. Shiztiko Mivachi.
Literature Cited
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chloroplasts. Polyphenol oxidase in Bc/a zn/pris.
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2. AVRON, NI. 1960. Photophosphorylation lv, swis.chard chloroplasts. Biochem. Biophy s. Acta 40:
257-72.
3. BRAY, G. A. 1960. A simple efficient liquid scintillator for couinting aqueouls solutions in a, liqui(d
scinitillationi cotunter. Anial. Biochem. 1: 279-85.
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URIBE AND JAGENDORF-LOCALIZATION OF ACIDS IN CHLOROPLASTS
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9. PACKER, L., D. XVr. DEAMER,
XV.
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