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 Downloaded from on June 15, 2017 - Published by www.plantphysiol.org Copyright © 1984 American Society of Plant Biologists. All rights reserved. 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 ). 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 .15pm nylon s M mesh [email protected]@*S pI P ,Ag/ml 0-2°C). Downloaded from on June 15, 2017 - Published by www.plantphysiol.org Copyright © 1984 American Society of Plant Biologists. All rights reserved. 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; ) 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) Downloaded from on June 15, 2017 - Published by www.plantphysiol.org Copyright © 1984 American Society of Plant Biologists. All rights reserved. 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. Downloaded from on June 15, 2017 - Published by www.plantphysiol.org Copyright © 1984 American Society of Plant Biologists. All rights reserved. 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 photosynthetic oxygen evolution by isolated chloroplasts. New Phytol 71: 201-255 5. DRY IB, JT WISKICH 1982 Role of the external adenosine triphosphate/ adenosine diphosphate ratio in the control of plant mitochondrial respiration. Arch Biochem Biophys 217: 72-79 6. GOLLER M, R HAMPP, H ZIEGLER 1982 Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat. Effect of electron transfer inhibitors and uncouplers. Planta 156: 255-263 7. HAMPP R 1980 Rapid separation of the plastid, mitochondrial and cytoplasmic fractions from intact leaf protoplasts of Avena. Planta 150: 291-298 1021 8. HAMPP R, M GOLLER, H ZIEGLER 1982 Adenylate levels, energy charge, and phosphorylation potential during dark-light and light-dark transition in chloroplasts, mitochondria, and cytosol of mesophyll protoplasts from Avena saiiva L. Plant Physiol 69: 448-455 9. HAMPP R, H ZIEGLER 1980 On the use ofAvena protoplasts to study chloroplast development. Planta 147: 485-494 10. HEBER U 1974 Metabolite exchange between chloroplasts and cytoplasm. Annu Rev Plant Physiol 25: 393-421 11. HEBER UW, KA SANTARIUS 1965 compartmentation of pyridine nucleotides in relation to photosynthesis. Biochim Biophys Acta 109: 390-408 12. HEBER U, KA SANTARIUS 1970 Direct and indirect transfer of ATP and ADP across the chloroplast envelope. Z Naturforsch 25b: 718-728 13. HEBER U, KA SANTARIUS, MA HUDSON, UW HALLIER 1967 Untersuchungen zur intrazellularen Verteilung von Enzymen und Substraten in der Blattzelle. I. Intrazellularer Transport von Zwischenprodukten der Photosynthese im Photosynthese-Gleichgewicht und im Dunkel-Licht-Dunkel-Wechsel. Z Naturforsch 22b: 1189-1199 14. HELDT HW 1966 The participation of endogenous nucleotides in mitochondrial phosphate-transfer reactions. Biochim Biophys Acta 7: 51-63 15. HELDT HW 1976 Transport of metabolites between cytoplasm and the mitochondrial matrix. In CR Stocking, U Heber, eds, Transport in Plants III. Springer-Verlag, New York, pp 235-254 16. KATO T, SJ BERGER, JA CARTER, OH LOWRY 1973 An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases. Anal Biochem 53: 86-97 17. KRAUSE GH, U HEBER 1976 Energetics of intact chloroplasts. In J Barber, ed, The Intact Chloroplast. Elsevier, North Holland Biomedical Press, pp 171214 18. LEHNER K, HW HELDT 1978 Dicarboxylate transport across the inner membrane of the chloroplast envelope. Biochim Biophys Acta 501: 531-544 19. LILLEY McCR, M STITT, G MADER, HW HELDT 1982 Rapid fractionation of wheat leaf protoplasts using membrane filtration. The determination of metabolite levels in chloroplasts, cytosol, and mitochondria. Plant Physiol 70: 965-970 20. LOWRY OH, JV PASSONNEAU 1972 A Flexible System of Enzymatic Analysis. Academic Press, New York 21. MUTO S, S MIYACHI, H USUDA, GE EDWARDS, JA BASSHAM 1981 Lightinduced conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide phosphate in higher plant leaves. Plant Physiol 68: 324-328 22. SELLAMI A 1976 Evolution des adenosine phosphates et de la charge energetique dans les compartiments chloroplastique et non-chloroplastique des feuilles de ble. Biochim Biophys Acta 423: 524-539 23. SIEs H 1982 Nicotinamide nucleotide compartmentation. In H Sies, ed, Metabolic Compartmentation. Academic Press, New York, pp 205-231 24. SOBOLL S, R ELBERS, R SCHOLZ, HW HELDT 1980 Subcellular distribution of di- and tricarboxylates and pH gradients in perfused rat liver. Z Physiol Chemie 361: 69-76 25. STITT M, W WIRTZ, HW HELDT 1980 Metabolite levels during induction in the chloroplast and the extrachloroplast compartments of spinach protoplasts. Biochim Biophys Acta 593: 85-102 26. STROTMANN H, S MURAKAMI 1976 Energy transfer between cell compartments. In CR Stocking, U Heber, eds, Transport in Plants III. Springer-Verlag, New York, pp 398-416 27. TAGER JM, RJA WANDERS, AK GROEN, W KUNZ, R BOHNENSACK, U KUSTER, G LETKO, G BOHME, J DuSZYNSKI, L WOJTCZAK 1983 Control of mitochondrial respiration. FEBS Lett 151: 1-9 28. TAKAHAMA U, M SHIMizu-TAKAHAMA, U HEBER 1981 The redox state of the NADP system in illuminated chloroplasts. Biochim Biophys Acta 637: 530539 29. URBACH W, MA HUDSON, W ULLRICH, KA SANTARIUS, U HEBER 1965 Verteilung und Wanderung von Phosphoglycerat zwischen den Chloroplasten und dem Cytoplasma waihrend der Photosynthese. Z Naturforsch 20b: 890-898 30. WISKICH JT 1977 Mitochondrial metabolite transport. Annu Rev Plant Physiol 28: 45-69 31. WISKICH JT 1980 Control of the Krebs cycle. In DD Davies, ed, The Biochemistry of Plants, Vol 2. Academic Press, New York, pp 243-278 Downloaded from on June 15, 2017 - Published by www.plantphysiol.org Copyright © 1984 American Society of Plant Biologists. All rights reserved.
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