Determination of Compartmented Metabolite Pools by a

Plant Physiol. (1984) 75, 1017-1021
0032-0889/84/75/1017/05/$0 1.00/0
Determination of Compartmented Metabolite Pools by a
Combination of Rapid Fractionation of Oat Mesophyll
Protoplasts and Enzymic Cycling'
Received for publication February 2, 1984 and in revised form April 9, 1984
RUDIGER HAMPP*, MARION GOLLER, AND HELENE FULLGRAF
Institut fur Biologie I, Universitat Tubingen, A uf der Morgenstelle 1, D-7400 Tuibingen I (R. H.); and
Institut fur Botanik, Technische Universitat Munchen, Arcisstrasse 21, D-8000 Mfinchen 2, Federal
Republic of Germany (M. G., H. F.)
ABSTRACI
In vivo pool sizes of a range of metabolites have been determined in
subcellular fractions of darkened and illuminated mesophyll protoplasts
of Avena sativa L. These estimations were made by combining a method
of rapid protoplast fractionation with enzymic cycling techniques. Results
are given for reduced and oxidized pyridine nucleotides, triose phosphates, 3-phosphoglycerate, inorganic phosphate, aspartate, malate, oxaloacetate, glutamate, 2-oxoglutarate, and citrate, from chloroplasts,
mitochondria, and a fraction representing the remainder of the protoplast.
The results indicate distinct differences of compartmented levels of
certain metabolites between darkened and illuminated protoplasts.
Cells of green tissue have two major systems for energy production, viz. that in chloroplasts and that in mitochondria. In
the dark, ATP formed by oxidative phosphorylation is exported
from mitochondria to energy consuming sites in the cell, while
a back flow of ADP maintains phosphorylation (15). Under
illumination there is additional production of ATP by chloroplasts: the latter exceeds considerably that by mitochondria (26).
From metabolite determinations on nonaqueously fractionated leaf tissue, Heber and Santarius (12) suggested that mitochondrial respiration of green tissue possibly is inhibited in the
light. This hypothesis was deduced from an observed increase in
the extrachloroplast ATP/ADP ratio after the onset of illumination, and invoked to the operation of the TP2/PGA shuttle
across the inner chloroplast membrane (10, 12). Such an increase
of the ATP/ADP ratio also has been observed by other authors
(8, 22). In addition, Goller et al. (6) showed that the incubation
of oat mesophyll protoplasts with permeable and specific inhibitors of the light-dependent electron transport is almost without
effect on the cytosolic adenylate ratio in the light. These results
were taken as evidence that light has no direct effect on oxidative
phosphorylation and that, potentially, mitochondria are equally
active under darkness and light.
Recent experiments with isolated animal and plant mitochondria (5, 27) indicate that a change in the external ATP/ADP
' Supported by a grant from the Deutsche Forschungsgemeinschaft
(Ha 970/6-7). Dedicated to Prof. H. Ziegler on the occassion of his 60th
birthday.
2Abbreviations: TP, triose phosphate; OAA, oxaloacetate; PGA, 3phosphoglycerate.
ratio is not the primary factor in the control of respiration in
isolated mitochondria. Therefore, Dry and Wiskich (5) concluded that, if leaf mitochondrial respiration is inhibited in the
light, the ATP/ADP ratio would be of minor importance in
regulation, i.e. respiration may be controlled in more than one
way (27).
Information on in vivo pool sizes of metabolites in various
compartments is limited and mainly has been derived from
nonaqueous fractionations of leaf tissue into chloroplast and
extrachloroplast material (e.g. Heber and coworkers [11-13,
29]). A better insight into mechanisms regulating mitochondrial
respiration is only possible, however, when pool sizes of a wider
range of metabolites within different cellular compartments are
known. Importantly, knowledge of how environmental perturbations affect the pool sizes is necessary.
In the present report we have combined our method of rapid
fractionation of mesophyll protoplasts (6, 7) with very sensitive
techniques for metabolite analysis (enzymic cycling: 20). This
approach enabled us to conduct in parallel assays of pyridine
nucleotides (reduced or oxidized), sugar phosphates, organic
acids, amino acids, and Pi in addition to adenylates.
The results indicate only limited metabolic alterations of compartmented pool sizes during fractionation, show good recoveries
starting from absolute amounts of 1 pmol of metabolite, and
compare compartmented pool sizes in darkened and illuminated
protoplasts.
MATERIALS AND METHODS
Plant Material and Culture Conditions. Seedlings of Avena
sativa L. (cv Arnold) were grown in hydroponic culture for 7 d.
Illumination (about 9 wm 2: Osram HQLS, 400 w + 2 Osram
concentra PAR spots, 75 w each) was started after 4 d of
germination in the dark (26°C, 80% RH).
Isolation of Protoplasts. Enzymic isolation and purification of
protoplasts from 0.5- to 1-mm-wide leaf segments was carried
out as reported earlier (6, 9). Protoplast numbers were counted
using a Neubauer double haemocytometer.
Incubation of Protoplasts. Protoplasts, resuspended in 0.5 M
sorbitol, 7.5 mM CaCl2, 5 mM KHCO3, and 25 mm Tricine (pH
7.6), were incubated in a Clark-type oxygen electrode (4) at 20°C.
Illumination was provided by two opposing 250 w slide projectors, yielding about 600 wm-2 at the surface of the electrode
vessel.
Fractionation of Protoplasts. The incubation of protoplasts
was terminated by centrifugal filtration, under the same conditions as that prevailing during incubation (i.e. illumination or
darkness). Aliquots (50 gl) of the protoplast suspension were
1017
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HAMPP ET AL.
1018
pipetted separately into 400-,ul centrifuge tubes that contained a
15-Am nylon mesh and several hydrophobic and hydrophilic
layers. The lowermost layer comprised 0.6 M sucrose and either
0.1 N NaOH (for reduced pyridine nucleotides) or 0.1 N HCI
(other metabolites) in 0.6 M sucrose. For preparation and handling of these microgradients see Goller et al. (6). As shown in
Figure 1, this integrated system of protoplast homogenation and
fractionation delivers three fractions (P; M; S: chloroplasts; mitochondria; supernatant, remainder of the cell) in addition to the
separate filtration of intact protoplasts (P', S'; see also "Results"
and legend to Fig. 1). Procedures for the correction of cross
contamination have been given in detail (6).
In preceding papers we also assessed possible alterations of
metabolite pools during this fractionation procedure, using adenylates, which have a high rate of turnover, as an example. There
we showed that metabolic reactions are terminated within less
than 2 s in pellet (chloroplasts) and supernatant fractions (mainly
cytosol) and obtained the amount of metabolites located in
mitochondria by subtracting metabolite pools associated with
chloroplasts and cytosol from those found in intact protoplasts
(6, 8). For reasons of comparison, we also used the more rapid
procedure of protoplast fractionation reported by Lilley et al.
(19). Using oat mesophyll protoplasts, both fractionation procedures, oil and membrane filtration, yielded comparable results.
Because of advantages in handling short periods of incubation
(time course experiments) and a less complicated correction for
cross-contamination, we preferred the technique of oil filtration.
Determination of Metabolites. All metabolites except pyridine
nucleotides were determined by reactions which were enzymically coupled to the oxidation or reduction of pyridine nucleotide. These assays were from Lowry and Passonneau (20; 'specific
step'). The amount of a product of this step was amplified by
enzymic cycling. For pyridine nucleotides, the neutralized samples were used for enzymic cycling without further treatment.
L.J
Plant Physiol. Vol. 75, 1984
Immediately after centrifugal filtration the centrifuge tubes
were transferred to degassed liquid nitrogen and kept at -80°C.
For analysis, the different fractions were obtained by cutting the
frozen tubes at the indicated positions (Fig. 1). Subsequently, the
fractions were thawed in ice-cold 0.1 N HCI (four parallels/1.5
ml; except reduced pyridine nucleotides). After vigorous shaking,
the extracts were kept at 100C for 5 min, immediately cooled
down on ice, and centrifuged at 8000g for 5 min. Aliquots of the
supernatant (1.0 ml) were brought to pH 6.5 by adding 1 N
KOH, and then stood on ice. Neutralization was carried out
shortly before starting the specific step (see below). One Ml (Pi),
5 Ml (TP) or 10 Ml (all other metabolites except pyridine nucleotides) of neutralized extract was assayed in a total volume of 25.0
Ml, including 5 ,l of 5 x concentrated specific step reagent and
double-distilled H20. Standards (about 25 pmol) were added in
a volume of 1.0ML.
In general, all assays were run in duplicate, and all determinations of metabolites were run at the same time with neutralized
aliquots from the same sample. Thus, for any experiment, the
reported levels of metabolites given are derived from the same
protoplast fractionation and are not stemming from different
incubations.
Specific Reagents. These reagents were S x concentrated (analytical enzymes from Boehringer, Mannheim, F.R.G.). If not
dialyzed, the enzymes were pelleted and dissolved in 25 mm Tris,
pH 8.1, 0.02% (w/v) BSA.
PGA: 250 mm imidazole-HCl (pH 7.0), 250 AM NADH, 6 mM
ATP, 5 mM MgCl2; 50 ug/ml PGA kinase, 250 ,gg/ml glyceraldehyde phosphate dehydrogenase. The commercial enzyme suspension had been dialyzed 2 x 90 min against Tris-HCl (25 mM,
pH 8.1) before incorporation into the reagent.
TP: 250 mm imidazole-HCl (pH 7.0), 250 AM NADH; 25 Ag/ml
TP isomerase, 50 Ag/ml glycerophosphate dehydrogenase (both
enzymes had been dialyzed, see above).
OAA: 250 mM imidazole-HCl (pH 7.0), 250 Mm NADH, 2Ag/ml
malate dehydrogenase.
Malate: 250 mM 2-amino-2-methylpropanol (pH 9.9), 5 mM
NAD, 200 mm glutamate; 25 jig/ml malate dehydrogenase, 20
Mg/ml glutamate-OAA-transaminase.
FIG. 1. Schematic presentation of the fractions obtained after centrifugation of the microgradients. After adding the protoplast suspension,
Glutamate: 250 mM Tris-acetate (pH 8.4), 1 mm NAD, 500 Mm
ADP; 500,g/ml glutamate dehydrogenase.
Citrate: 250 mm imidazole-HCl (pH 7.0), 250 Mm NADH, 200
Mm ZnC12, 1 Mg/ml malate dehydrogenase; 400 Mg/ml citrate
lyase.
Aspartate: 250 mM imidazole-HCl (pH 7.0), 250 AM NADH, 1
mM oxoglutarate; 1 Ag/ml malate dehydrogenase, 40 Mg/ml
glutamate-OAA-transaminase.
Oxoglutarate: 250 mM imidazole-acetate (pH 7.0), 125 mm NH4
acetate, 500 Mm ADP, 250 AM NADH; 2 Mg/ml glutamate dehydrogenase.
Pi: 250 mM imidazole-HCI (pH 7.0), 80 mg/ml glycogen (containing 60 mg BSA and dialyzed against 25 mM acetate buffer,
pH 4.6), 5 mm NADP, 50 MM 5' AMP, 5 mM EDTA, 2.5 mM
Mg-acetate, 2.5 mM DTT, 2.5 Mm glucose-1,6-diphosphate; 10
G-6-P dehydrogenase, 50 Ag/ml phosphoglucomutase
(both enzymes were washed three times in 3.2M (NH4)2S04),
250OMg/ml phosphorylase a (washed with double-distilled H20 at
both types of gradients are covered with punctured caps, containing 5 l
of 0.3 N HCI or Tris-medium (0.5 Tris-HCI, pH 7.6; 1 KCI, 50 mM
MgCl2; see [8]). In the gradient tube with the inserted nylon mesh,
protoplasts are ruptured during centrifugation and the resulting homogenate forms three aqueous fractions (P, pellet (chloroplasts); M, middle
fraction (mainly mitochondria); S, supernatant [mainly cytosol]) which
are separated by layers of silicone oil. The second tube (no net) filtrates
intact protoplasts through silicone oil and delivers two fractions: P',
pellet of intact protoplasts;S', supernatant, material released from broken
protoplasts (for details see 6, 7).
Reaction blanks were obtained by adding 5 Ml1 N HCI (PGA,
TP, OAA, citrate, aspartate, oxoglutarate) or 5 Ml 1 N NaOH
(malate, glutamate, Pi) to the specific reagent before including
the sample aliquot. All assays (blanks, samples without and with
internal standards) were run at 25°C for 15 to 30 min and
terminated as described above for the blanks. Completion of the
reactions was always checked by running identical assays on a
macro level with standards in the photometer. After termination,
all samples were kept at100C for 5 min and then immediately
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nylon
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[email protected]@*S
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0-2°C).
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1019
COMPARTMENTED METABOLITE POOLS
cooled on ice.
Enzymic Cycling. To measure accurately the small amounts
of NAD (PGA, TP, OAA, citrate, aspartate, oxoglutarate),
NADH (malate, glutamate), NADP, and NADPH (Pi), the technique of enzymic cycling was employed (see 16, 20 for details).
For this purpose, 2-IA aliquots of the terminated assays were used
without neutralization and added to 25 1l of double strength
cycling reagent in a total volume of 50 ,l.
For the determination of oxidized pyridine nucleotides, the
neutralized (pH 6.5) HCI extract was directly used for cycling
(25-01 aliquots).
Reduced pyridine nucleotides were determined in 25-,gl aliquots of neutralized 0.1 N KOH extracts (pH 7.8), immediately
after finishing the protoplast fractionation procedure, as repeated
freezing and thawing (see acidic extracts) led to a significant
degradation of reduced pyridine nucleotides already after 1 d.
recovery was high in fractions P, M, and P'. However, recovery was lower in the supematants S and S'. This
oxidation appears to be due to the effect of vacuolar constituents
which are released during homogenization (protoplasts pass a
1 5-jim nylon mesh during centrifugation) or result from broken
protoplasts,
material contained in the protoplast suspension before centrifugal filtration (S'). The loss of NADH and NADPH could not be
diminished by adding reducing agents (e.g. 10 mm isoascorbate)
or by increasing the buffer strength. For this reason, levels of
reduced pyridine nucleotides contained within the cytosol (Table
II) were calculated by subtracting the amount recovered in P
(chloroplasts) and M (mainly mitochondria) from P' (intact
filtered protoplasts). An error as large as 20% is possible for these
derived values.
Compartmented Metabolite Levels. In Table II the compartmented pool sizes of all metabolites in the dark (7 min dark,
20C) are compared with those in the light (5 min illumination,
RESULTS
20°C).
Upon illumination, the level of TP increases in both the
Recovery of Metabolites. To determine the extent to which chloroplast
stroma and the cytosol, whereas that of PGA declines
metabolite pool sizes were altered during frationation, extraction
the stroma, staying fairly constant within the cytosol. Surprisand assay, protoplasts were fractionated in the absence and in
there were also considerable amounts of both metabolites
presence of metabolite standards. For this purpose, the individual ingly,
be considaqueous layers of the gradient (and the droplet of Tris-medium associated with mitochondria. This observation may
the
which
has
not been
as
a
contamination
ered
cytosol
by
injected into the protoplast suspension during centrifugation;
[6]) were mixed with standard solutions. In Table I, the recovery corrected for (intermembrane space); however, such an argument
of metabolites is given as per cent of the added quantity (0.03-1 would only apply for TP (mitochondrial content -20% of cytonmol). Recovery was approximately 100% for aspartate, malate, sol) but would not be acceptable for PGA (mitochondrial level
glutamate, Pi, oxoglutarate, citrate, NAD, and NADP in all higher than cytosol), aspartate, and citrate (in both cases mitogradient fractions. Recovery of TP and PGA was high in fractions chondrial levels are far below those found in the cytosolic/
P and P' (chloroplasts and filtered protoplasts, respectively), but vacuolar compartment). Thus, if these compounds do not behave
that in the middle fraction (M: about 80% mitochondria <10% differently with regard to their adsorption to cellular membranes
chloroplasts) was lower. Recovery in the supematant of both or location in the intermembrane space, there are indeed considfractionated (S) and oil filtered (S') protoplast suspensions was erable amounts of TP and PGA associated with mitochondria
high. The recovery of OAA was relatively poor, with best results from oat mesophyll protoplasts.
Aspartate concentration is higher in chloroplasts and cytosol/
in P, P', and S'.
Problems, which we could not solve, were encountered in the vacuole from illuminated protoplasts than in the dark. This
assay for NADH and NADPH. In the absence of protoplasts compound was not detectable in mitochondria regardless of
(NADH and NADPtI standards were added to gradient fractions conditions (i.e. c0.05 nmol/106 protoplasts).
There was also an increase of stromal and cytosolic/vacuolar
and centrifugation as well as all other procedure carried through
as in the case of a protoplast fractionation), the recovery was malate upon illumination. The level in mitochondria was comsimilar to that of the other compounds. In the presence of parable to that found in chloroplasts, but was about the same in
Table I. Recovery of Metabolites
Aliquots of a mixture of metabolite standards were added to all aqueous gradient layers before starting
protoplast fractionation. Recovery: ([metabolite in protoplast fractions with added standards] - [metabolite in
protoplast fractions with no standards added]): (chemical amount of standard added) x 100 (%). The values
given are the mean of 5 experiments. The SE was less than 10% except for metabolites marked with an asterisk.
Numbers in parentheses indicate recovery of standard metabolites after fractionation without protoplasts. For
the fractions designed P, M, S, S', P' see Figure 1.
Fraction
Metabolite Standards
5'
M
P
P
S
TP*
PGA
Aspartate
Malate
Glutamate
Pi*
OAA*
Oxoglutarate
Citrate
NAD
NADP
NADH*
NADPH*
111
100
87
80
84
106
79
94
90
119 (99)
101 (107)
92 (103)
112 (92)
67
66
92
87
92
103
30
107
107
86 (92)
84 (90)
86 (88)
105 (115)
152
77
100
78
81
137
49
115
105
97 (103)
84 (92)
10 (73)
7 (84)
173
130
115
82
89
130
79
115
99
92 (101)
79 (112)
20 (71)
4 (77)
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120
140
92
110
83
116
113
97
106
127 (105)
119 (99)
57 (110)
128 (92)
1020
HAMPP ET AL.
Metabolite
Plant Physiol. Vol. 75, 1984
Table II. Levels of Metabolites in Subcellular Compartments ofOat Mesophyll Protoplasts'
Cytosol + Vacuole
Mitochondria
Chloroplasts
Dark
Light
Light
nmol/106 protoplasts
64.0 (20.1)
88.0 (14.4)
2.8 (14.0)
2.2 (21.4)
6.1 (53.0)
3.6 (22.6)
<0.05
<0.05
1.0 (2.6)
0.80 (4.4)
2.0 (29.4)
2.8 (12.6)
0.25 (18.1)
0.20 (10.7)
<0.05
<0.05
Dark
Dark
Light
246.0 (77.4)
475.0 (77.5)
7.9 (2.5)
50.0 (8.2)
Pi
13.0 (65.0)
7.0 (68.0)
4.2 (21.0)
1.1 (10.7)
TP
1.1
(9.6)
0.80 (5.0)
4.3 (37.4)
11.5 (72.3)
PGA
12.5 (75.3)
3.9 (66.1)
4.1 (24.7)
2.0 (33.9)
Aspartate
35.0 (92.3)
16.5 (90.2)
1.9 (5.0)
1.0 (5.5)
Malate
1.7 (25.0)
8.0 (35.9)
3.1 (45.6)
11.5 (51.6)
Glutamate
1.0 (71.0)
1.3 (67.9)
0.15 (10.9)
0.40 (21.4)
Oxoglutarate
10.5 (100.0)
8.5 (100.0)
<0.05
<0.05
Citrate
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
OAA
1.20 (59)
1.1 (58.2)
0.35 (19.2)
0.41 (22.5)
0.40 (26.8)
0.42 (28.2)
NAD
0.15 (22)
0.35 (37.2)
0.19 (20.2)
0.14 (11.4)
0.40 (42.6)
0.38 (30.9)
NADH
0.18 (50.0)
0.12 (33.3)
<0.05
0.24 (66.7)
<0.05
0.18 (50.0)
NADP
0.65 (80.2)
0.73 (89.0)
<0.05
0.09 (11.0)
<0.05
0.16 (19.8)
NADPH
a Values in parentheses: % of total; for comparison with other data: 106 protoplasts are equivalent to about 100 ug of Chl; volumes (j.1/106
protoplasts): protoplasts, 23; chloroplasts, 2.5; mitochondria, 1.0. The values are means of 3 independent experiments; the SE was between 5 and
20%. The average rate of photosynthesis was 84 Oimol 02 evolved-mg-' Chl- h-' when aliquots of the protoplast suspension were taken for
fractionation.
and nonaqueously isolated chloroplasts (11, 28), Heber and
Santarius (1 1) reported very low NADH/NAD ratios for chloroplasts and an extrachloroplast fraction (NADH between 2 to
5% of the total NAD(H) system in both light and darkness).
Our data, like that of Takahama et al. (28), indicate no lightmediated interconversion of diphospho- into triphosphopyridine
nucleotides as reported by Muto et al. (21).
Chloroplast-associated pool sizes of TP, PGA, Pi show typical
differences between darkened and illuminated protoplasts (Table
II) as reported by others (13, 25, 29). From labeling experiments
and pool size determinations, Heber et al. (13) and Urbach et al.
(29) calculated about 75% of the total PGA and about 20 to 25%
of total dihydroxyacetone phosphate to be chloroplast associated.
This compares well with about 70 and 21% (TP), respectively,
in our fractionation study, as can be calculated from Table II.
The concentration of OAA has been reported to be between
10 and 20 uM in spinach leaves (17), equivalent to about 0.2
nmol/106 oat mesophyll protoplasts (about 23 ,l/1O6 protoplasts;
see 8). As such a value is near the detection limit of our methods,
this could explain why we had problems trying to demonstrate
an association of OAA with subcellular fractions.
Anderson and Walker (1) showed that illuminated intact pea
chloroplasts support (NH4+ + oxoglutarate)-dependent 02 evolution, without the requirement of exogenous glutamate. This
was attributed to a high endogenous concentration of glutamate.
The glutamate concentration (6 mM; volume of the stroma of
oat chloroplasts: 2.5 ,A/106 protoplasts; see (8]) can be calculated
to be sufficient to account for the rates observed by them.
From results given by Lehner and Heldt (18) on aqueously
isolated spinach chloroplasts, higher glutamate as well as malate
DISCUSSION
and aspartate concentrations can be calculated in comparison to
With respect to stromal levels of metabolites, some of the our results; but the data are not directly comparable: isolated
results given in this paper agree quite well with those obtained chloroplasts are subject to osmotically induced changes in volby nonaqueous fractionation of lyophilized leaves (1 1, 12, 29). ume due to the isolation procedure.
As far as we know, an association of citrate with chloroplasts
For pyridine nucleotides, Heber and Santarius ( 11) reported less
than 50% of the total NADPH and more than 50% of NADP to has not been reported.
Data given in the literature on compartmentation of metabobe chloroplast associated. In addition, 25% of the NADP(H)
system of chloroplasts from darkened cells, and 70 to 100% of lites in green plant cells only distinguish between chloroplasts
the extrachloroplast space were in the reduced state. These values and extrachloroplast space, i.e. the remainder of the cell. Our
results differentiate between mitochondrial and cytosolic/vacuare similar to our results (Table II). Upon illumination (5 min),
olar pool sizes of a wider range of metabolites. Metabolite levels
we find a lower level of NADPH (due to an intense CO2 fixation;
compare 28). While the results on the NADP system are com- associated with mitochondria from oat mesophyll protoplasts are
parable to reported ratios in the light steady state of aqueously to some extent comparable to results given for animal mitochon-
light and darkness.
Glutamate was also fairly constant in mitochondria. Stromal
and cytosolic/vacuolar levels, however, were lower in the light
than in the dark.
Except in the extraorganellar space, levels of oxoglutarate were
quite low in both stroma and matrix; light-dependent changes
were not detected.
Citrate was only detectable in the cytosolic/vacuolar compartment and was slightly higher there if protoplasts had been illuminated.
The level of Pi strongly responded to a dark-light change in all
compartments; its decrease was larger in the chloroplast stroma,
which is interpreted to be a result of starting photosynthesis and
subsequent Pi transport and consumption.
The analysis of pyridine nucleotide compartmentation indicates that NADP(H) is obviously not located in mitochondria
from oat mesophyll protoplasts with levels below the detection
limit (50 pmol/106 protoplasts).
In general, the sum of oxidized and reduced forms of each,
NAD and NADP, was the same in the light and in the dark.
In the cytosol, NAD was lower in the dark compared to 5 min
illumination, whereas chloroplasts and mitochondria had similar
steady state levels under both conditions. In contrast, the stromal
NADP level showed the opposite trend, with a lower NADPH/
NADP ratio in the light, which is interpreted to be due to a high
demand to reducing equivalents for CO2 fixation (NADPH as
limiting factor). On the other hand, the redox state of the
cytosolic NADP system appeared to be higher in the light compared with dark treated cells.
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COMPARTMENTED METABOLITE POOLS
dna. As in oat, mitochondria from rat liver contain between 20
and 50% of the total cellular NAD(H) system, with the oxidized
form dominating (23). Compartmentation of the NADP(H) system, however, is quite different. Triphospho-pyridine nucleotides
were below our detection limit in oat mitochondria, whereas in
rat liver cells the NADP(H) system is predominantly located in
these organelles (23).
Similarly, the concentrations of aspartate and citrate were
below our detection limit in oat mitochondria. These data also
are in contrast to the results on rat liver mitochondria, where
both metabolites are mainly matrix located (24). In oat mesophyll
protoplasts the compartmentation is exactly opposite, with all of
the citrate and most of the aspartate being located outside
chloroplasts and mitochondria (possibly in the vocuole). However, the bulk of the cellular citrate is synthesized in mitochondria because citrate synthase is primarily located here (2). From
studies on animal mitochondria (31) there is evidence that the
tricarboxylate carrier transports citrate from the mitochondria to
the cytoplasm in exchange for malate, which is present at high
concentrations in the cytosolic/vacuolar compartment (Table
II). Furthermore, malate is oxidized much more rapidly by
mitochondria than citrate is; thus the malate/citrate exchange
may, by preference, export citrate, which explains the low citrate
level associated with oat mitochondria.
To our knowledge there is only one report on the location of
PGA in rat liver mitochondria (14) and no translocation of PGA
or TP has been investigated (see 30, 31), although it has been
suggested that, in animal cells, dihydroxyacetone phosphate has
some function in translocating cytosolic protons across the mitochondrial membrane (3). As these two metabolites show considerable changes in their pool sizes during a dark/light transition, their association with oat mitochondria could be quite
interesting with regard to the regulation of mitochondrial respiration in darkness and light, in addition to the adenylate (8) and
the pyridine nucleotide systems (see 31).
Acknowledgment-We gratefully acknowledge the thorough introduction into
enzymic cycling techniques by Prof. W. H. Outlaw, Jr., Florida State University.
LITERATURE CITED
1. ANDERSON JW, DA WALKER 1983 Ammonia assimilation and oxygen evolution by a reconstituted chloroplast system in the presence of 2-oxoglutarate
and glutamate. Planta 159: 247-253
2. BIRNBERG PR, DL JAYROE, JB HANSON 1982 Citrate transport in corn mitochondria. Plant Physiol 70: 511-516
3. BUCHER T, M KLINGENBERG 1958 Wege des Wasserstoffs in der lebendigen
Organisation. Angew Chemie 70: 522-570
4. DELIEU T, DA WALKER 1972 An improved cathode for the measurement of
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