CHEN TM, P DIITRICH, WH CAMPBELL, CC BLACK 1974 Metabolismof ... HERBERT M, C BURKHARD, C SCHNARRENBERGER 1979 A survey for isoen-.
Plant Physiol. (1986) 80, 301-304 0032-0889/86/80/0301/04/$0 1.00/0
Distinction between Cytosol and Chloroplast FructoseBisphosphate Aldolases from Pea, Wheat, and Corn Leaves Received for publication May 14, 1985 and in revised form September 12, 1985
CLAUS SCHNARRENBERGER* AND INGO KRUGER'
Institutfiur Pflanzenphysiologie und Zellbiologie, Freie Universitat Berlin, Fachbereich Biologie, D-1000 Berlin 33 (West) ABSTRACT A reinvestigation of cytosol and chloroplast fructose-1,6-bisphosphate (FBP) aldolases from pea (Pisum sativum L.), wheat (Triticum aestivum L.) and corn leaves (Zea mays L.) revealed that the two isoenzymes can be separated by chromatography on diethylaminoethyl (DEAE)-cellulose although the separation was often less clear-cut than for the two aldolases from spinach leaves. Definite distinction was achieved by immunoprecipitation of the two isoenzymes with antisera raised against the respective isoenzymes from spinach leaves. The proportion of cytosol aldolase as part of total aldolase activity was 8, 9, 14, and 4.5% in spinach (Spinacia oleracea L.), pea, wheat, and corn leaves, respectively. For corn leaves we also obtained values of up to 15%. The K. (FBP) values were about 5-fold lower for the cytosol (1.1-2.3 micromolar concentration) than for the chloroplast enzymes (8.0-10.5 micromolar concentration). The respective Km (fructose-i-phosphate, FIP) values were about equal for the cytosol (1.0-23 millimolar concentration) and for the chloroplast aldolase (0.6-1.7 millimolar concentration). The ratio V (FIP)/V (FBP) was 0.20 to 0.27 for the cytosol and 0.07 to 0.145 for the chloroplast aldolase. Thus, cytosol and chloroplast aldolases from spinach, pea, wheat, and corn leaves differ quite considerably in the elution pattern from DEAEcellulose, in immunoprecipitability with antisera against the respective isoenzymes from spinach leaves, and in the affinity to FBP.
In green leaf cells, sugar phosphate metabolism takes place in the cytosol and in chloroplasts (e.g. 26). Thereby, the chloroplast envelope controls a limited exchange of sugar phosphates and other molecules either by diffusion or translocator mediated countercurrent exchange (9, 15). Since the first discovery of cytosol and chloroplast specific isoenzymes of sugar phosphate metabolism in the respective cell compartments (1), the view has developed that two isoenzymes exist for virtually all reactions of glycolysis, gluconeogenesis, and the oxidative pentose phosphate cycle, with one isoenzyme located in the cytosol and the other in chloroplasts (23). Just a few key enzymes of the Calvin cycle are present in the chloroplasts exclusively, e.g. ribulose bisphosphate carboxylase/oxygenase and ribulose-5-P kinase. While all pairs of isoenzymes of sugar phosphate metabolism differ in charge, many of them have very similar molecular sizes and catalytic properties. This may imply that they are different but very similar proteins. The ratter may apply particularly to the two aldolases of pea and corn which were reported to have virtually identical mol wt, substrate affinities, and terminal amino acid sequences (3, 4, 12). However, recent investigations
of the two spinach aldolases indicated rather profound differences between these two isoenzymes as deduced from their subunit mol wt, from the lack of extensive immunochemical crossreaction, from peptide analyses after tryptic digestion, and from terminal amino acid sequencing (17, 20). In leaves of wheat and corn, it was difficult to identify the cytosol aldolase by conventional methods (17, 22) so that even the existence of separate cytosol and chloroplast aldolases had to be questioned. For this reason we have applied an improved combination of ion-exchange chromatography and immunoprecipitation to identify the two pea, wheat, and corn aldolases. Quantitative estimates of the two aldolases in green leaves are reported. In addition we found large differences in the apparent affinity for FBP2 but not in the apparent affinity for FIP and in the V(Fl P)/ V(FBP) ratio for the two spinach, pea, wheat, and corn aldolases. MATERIALS AND METHODS
Materials. Spinach (Spinacia oleracea L., type Monopa), pea (Pisum sativum L., type Kleine Rheinlanderin), wheat (Triticum aestivum L., type Kolibri), and corn (Zea mays L., type Inrafruh) were grown from seeds in greenhouses. Leaves (or shoots in the case of pea) of well developed plants were harvested and used immediately for analyses. Separation of Cytosol and Chloroplast Aldolases. The two aldolases were separated by anion-exchange chromatography on DEAE-cellulose. About 20 g of tissue were homogenized for 1 min at 4C in 4- to 5-fold of grinding buffer (10 mm K-phosphate, pH 8.6, and 10 mM f3-mercaptoethanol) using a Waring Blendor. Corn leaves were homogenized for an additional minute with a Virtis "45" homogenizer in order to break the bundle sheath cells. The homogenates were squeezed through two layers of Miracloth and centrifuged for 12 min at 48,000g. The supernatant was readjusted to pH 8.6 and diluted with 10 mm f3mercaptoethanol, pH 8.6, to a final conductivity of 3 mS or less. The solution was immediately applied to 50 ml of wet packed DE32-cellulose (Whatman Lab. Sales, Springfield Mill, Maidstone, Kent/England) and equilibrated with grinding buffer on a funnel under suction. The resin was packed into a 3 cm wide glass column and proteins were eluted by a linear 200 ml gradient of 0 to 0.5 M KCI in grinding buffer overnight. Fractions of 3 ml were collected and assayed for aldolase activity (see below) and the KCI concentration measured by conductimetry. Assays and Immunoprecipitation of Aldolases. Fructose-bisphosphate aldolase (EC 4.1.2.13) was measured spectrophotometrically at 20°C in a standard assay by coupling the reaction to the glycerol-3-P dehydrogenase (NAD+) (EC 1.1.1.8) reaction according to Wu and Racker (29). The reaction mixture of 1 ml
' Present address: Botanisches Institut, Technische Universitait Darms2Abbreviations: FBP, fructose-1,6-bisphosphate; tadt, D-6 100 Darmstadt, Schnittspahnstraf3e, FRG. phosphate. 301
FIP,
fructose-l-
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contained 50 mm Tris-HCl, pH 7.5, 4.5 mM MgCl2; 1 mm EDTA; 1 unit each of triosephosphate isomerase and glycerol-3-P dehydrogenase; 250 gM NADH, and 1.0 mM FBP trisodium salt (Boehringer, Mannheim, FRG). Activity of FIP aldolase was measured accordingly except that Fl P disodium salt (Boehringer, Mannheim, FRG) was substituted for FBP. The enzyme activity of 1 unit is equivalent to the cleavage of 1 Mmol FBP (= 2 umol NADH) or F1P (= 1 Mmol NADH) per min. To distinguish between cytosol and chloroplast aldolases, one of the two aldolases was removed by immunoprecipitation before assaying enzyme activity. Antisera against the cytosol and chloroplast aldolase were prepared as described previously ( 17). For immunoprecipitation 20 Ml of an aldolase preparation and 20 Ml of antiserum were incubated for 1 min in an Eppendorf reaction vessel. Five ,l of a 12% (v/v) suspension of Staphylococcus aureus Cowan I cells (immunoprecipitin from Bethesda Research Laboratory, Bethesda, MD) were added to bind immune complexes to protein A on the surface of these cells. After centrifugation in a table top centrifuge for 1 min, the residual enzyme activity in the supernatant was determined in the standard assay.
RESULTS Separation of cytosol and chloroplast aldolases from a crude extract of pea, wheat, or corn by chromatography on DEAEcellulose often yields a single peak of aldolase activity. Only through high resolution (Fig. la-c) a small shoulder or peak preceding the main activity peak can be observed in the elution profile. Equivalent fractions in gradients with spinach leaf enzymes have been shown in the past to contain cytosol aldolase in a well separated, small peak preceding the main peak which contained chloroplast aldolase (17, 18). In the present study we have, therefore, removed one of the two aldolases by immunoprecipitation with specific antisera prior to the assay to distinguish the elution profiles of the two aldolases. Then, indeed, two independent, partly separated profiles become visible. During immunoprecipitation care was taken that only an amount of total aldolase was used that could be bound by an aliquot of antiserum. As determined with spinach leaf aldolases the antichloroplast-aldolase antiserum had less than 0.1% cross-reaction with the cytosol aldolase and the anti-cytosol-aldolase antiserum had 30% cross-reaction with the chloroplast aldolase of spinach. On this basis we conclude that the measured activity of the cytosol aldolase was entirely due to this isoenzyme and the measured activity of the chloroplast aldolase was fully due to this isoenzyme although reduced by one third or more because of coprecipitation by cross-reacting antibodies. A quantitative evaluation of the elution profiles of spinach, pea, wheat, and corn leaf aldolases reveals (Table I) that 8, 9, 14, and 4.5%, respectively, of the total aldolase activity in leaves of these plants is cytosolic and the remaining enzyme activity from chloroplasts. Occasionally with corn leaves we encountered values of up to 15% of total activity. These estimates appear to be fairly firm since the recovery of activity after chromatography on DEAE-cellulose is usually at least 85%. In addition the cytosol aldolase is from our experience far more stable than the chloroplast enzyme, thus emphasizing the low values for the cytosol aldolase. The cytosol and chloroplast aldolases show also significant differences in their kinetic properties. The substrate dependences for FBP and FlP follow strict Michaelis-Menten kinetics. This is documented for the two spinach leaf isoenzymes in Figure 2. The Km (FBP) values for the cytosol aldolase are 5 times smaller than for the chloroplast aldolase of spinach, pea, wheat, and corn (Table II). However, the Km (F1P) values and the ratio V (Fl P)/ V (FBP differ by only a factor of two or less.
Plant Physiol. Vol. 80, 1986
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FIG. 1. Resolution of total aldolase (0), cytosol aldolase (0), and chloroplast aldolase (0) of pea (a), wheat (b), and corn (c) leaves by ionexchange chromatography on DEAE-cellulose. A, KCI concentration.
DISCUSSION In the present study we have shown that the cytosol and chloroplast aldolases of pea, wheat, and corn can be separated by ion-exchange chromatography on DEAE-cellulose and behave similarly to the two aldolases from spinach leaves (17, 18, 20). Because the cytosol aldolase represents only a small proportion of total aldolase activity, the cytosol aldolase peak is often
CYTOSOL AND CHLOROPLAST ALDOLASES Table I. Relative Proportion ofCytosol and Chloroplast Aldolase in Spinach, Pea, Wheat, and Corn The respective proportions were calculated from enzyme distribution profiles as shown or similar to those shown in Figure 1. Aldolase
Spinach Pea Wheat Corn
Cytosol
Chloroplast
8 9 14 4.5
92 91 86 95.5
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FIG. 2. Lineweaver-Burk plots with FBP (a) and FIP (b) for the cytosol (0) and chloroplast aldolase (0) of spinach leaves. The maximal velocity with FBP was the same for all experiments.
Table II. Kinetic Properties ofCytosol and Chloroplast Aldolasesfrom Spinach, Pea, Wheat, and Corn Km and V values were obtained from Lineweaver-Burk plots of 2 to 4
independent experiments. Plant
Aldolase Chloroplast
Species
Cytosol
Km (FBP)
Spinach Pea Wheat Corn
2.3 1.6 1.1 2.0
Km (FIP)
Spinach Pea Wheat Corn
2.3 1.0 1.7
1.7 1.6 0.6
1.4
1.7
Spinach Pea Wheat Corn
0.27 0.20 0.20 0.25
M
10.3 9.1 8.3 8.0 mM
ratio V(FIP)/V(FBP)
0.09 0.10 0.145 0.07
considerably superimposed by the chloroplast aldolase peak, but can easily be uncovered by immunoprecipitation of the latter isoenzyme. Enrichment of cytosol aldolase can be achieved by rechromatographing on DEAE-cellulose (data not shown). Failure to separate the two isoenzymes in the past ( 17, 22) was probably due to insufficient resolution. This may have led to somewhat different conclusions. The elution properties of the two aldolases from pea, wheat, and corn indicate that the cytosol aldolase is the less negatively charged protein at pH 8.6. This is
303
in good agreement with the previous observation on the pea enzymes in which the isoelectric point of the cytosol aldolase is higher than the isoelectric point of the chloroplast aldolase (1, 7). The kinetic properties of cytosol and chloroplast aldolases from spinach, pea, wheat, and corn leaves differ in that the Km (FBP) of the cytosol aldolase is 5 times lower than that of the chloroplast aldolase. This is a new finding and requires full sensitivity of the assay down to 1 gmol per min. The respective Km values for Fl P differ by a factor of two or less. Since it is believed that the top carbon atoms one to three of FBP are the prime binding target for the substrate, it is proposed that there is a difference in the binding site for the carbon atoms four to six of the cytosol and chloroplast aldolases causing the different affinity for FBP. Summarizing the present view on the two isoenzymes, it seems that the difference in charge, the immunochemical difference, and the difference in substrate affinity for FBP appear to be consistent for all cytosol and chloroplast aldolases with respect to at least higher plants. The present data are in agreement in that the two aldolases are coded for by different gene loci (2, 27) and have a very different primary structure as revealed by immunochemical comparison and analysis of peptides after tryptic digestion of the two proteins (17, 19, 20). The quantitative estimation of cytosol and chloroplast aldolases indicates that the cytosol isoenzyme represents only 8, 9, 14, and 4.5%, respectively, of the total aldolase activity in spinach, pea, wheat, and corn leaves, as compared to the high proportion of the chloroplast enzyme. These values varied only within small limits in these tissues except for corn leaves. In the latter tissue we encountered values between 1 and 16% for the cytosol aldolase. The reason for this inconsistency remains unknown. Nevertheless, the values emphasize the previously proposed distinctiveness of two groups of isoenzymes of the sugar phosphate metabolism in the cytosol and chloroplasts of green leaf cells. Group one represents isoenzymes with only small proportions (7-15%) of cytosol isoenzymes and group two with high proportions (50-70%) of cytosol isoenzymes. Group one appears to contain those isoenzymes for which the chloroplast enzyme functions in the Calvin cycle and group two the isoenzymes for the other reaction of glycolysis, gluconeogenesis and the oxidative pentose phosphate pathway (I18). In leaves of the C4 plant corn, we could only resolve one major aldolase which, in a previous study by us, had been proposed to be the chloroplast enzyme present in bundle sheath cells (17). This proposal still holds for the major aldolase isoenzyme reported in the present study since it was identified as a chloroplast enzyme by immunochemistry and since in leaves of C4 corn plants most, if not all, aldolase activity is restricted to the bundle sheath cells (8, 13, 24). Failure to detect aldolase activity in mesophyll cells during previous cell fractionation studies was probably due to the fact that it represented only a small proportion of total activity. Although we do not have data on the cellular compartmentation of the newly identified cytosol aldolase from corn leaves, we would expect that it is located in mesophyll cells. This is based on the fact that sucrose synthesis in C4 plants is largely confined to mesophyll cells (6, 10, 21) and that sucrose synthesis has been shown to take place mainly in the cytosol (5, 28). However, the requirement of a cytosol aldolase in mesophyll cells for sucrose synthesis, but not for the operation of the oxidative pentose phosphate cycle (16), could be abandoned if a transport of glucose- l-P or amylose is assumed between bundle sheath and mesophyll cells (1 1, 25) in addition to the well documented triose phosphate exchange between the two cell types (14). From a physiological point of view, the question may be raised whether the chloroplast aldolase and the small amount of cytosol
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aldolase are sufficient to account for their presumed functions. Our extracted aldolases show at most activities of 1 to 2 units per g of fresh weight or 1 to 2 umol min-' g-' fresh weight. Average photosynthetic CO2 fixation rates may proceed at 100 to 200 gmol h-' mg-' Chl in C3 plants and twice as fast in C4 plants. If a conversion of 2 g fresh weight to 3 mg Chl is assumed (30) the total extracted aldolase activity is in the order of 40 to 80 ,umol h-' mg -'Chl. This aldolase activity would be just sufficient for its function in the Calvin cycle since it is required to at most one half the rate of photosynthetic CO2 fixation. The activity of the cytosol aldolase would then be roughly one-tenth of the total. This again would be at the lower end of the scale, however sufficient, in order to function in sucrose synthesis in the cytosol since only one-sixth of the CO2 fixation rates would be needed for aldolase activity in gluconeogenesis from triose phosphate in the cytosol. We are aware of many experimental and theoretical pitfalls in such a calculation, but attention should be focused on this problem since it has never been handled properly in the past. Acknowledgment-The authors thank Mrs. Renate Grubnau for excellent technical help. LITERATURE CITED 1. ANDERSON LE, VR ADVANI 1970 Chloroplast and cytoplasmic enzymes. Three distinct isoenzymes associated with the reductive pentose phosphate cycle. Plant Physiol 45: 583-585 2. ANDERSON LE, DA LEVIN 1970 Chloroplast aldolase is controlled by a nuclear gene. Plant Physiol 46: 819-820 3. ANDERSON LE, RL HEINRIKSON, C NoYEs 1975 Chloroplast and cytoplasmic enzymes. Subunit structure of pea leaf aldolases. Arch Biochem Biophys 169: 262-268 4. ANDERSON LE, I PACOLD 1972 Chloroplast and cytoplasmic enzymes. IV. Pea leaf fructose 1.6-diphosphate aldolases. Plant Physiol 49: 393-397 5. BIRD IF, MJ CORNELIUS, AJ KEYS, CP WHITTINGHAM 1974 Intracellular site of sucrose synthesis in leaves. Phytochemistry 13: 59-64 6. BUCKE C. IR OLIVER 1975 Location of enzymes metabolising sucrose and starch in the grasses Pennisettum putrpureum and Muhlenbergia montana. Planta 122: 45-52 7. BUKOWIECKI AD. LE ANDERSON 1974 Multiple forms of aldolase and triose phosphate isomerase in diverse plant species. Plant Sci Lett 3: 383-386 8. CHEN TM, P DIITRICH, WH CAMPBELL, CC BLACK 1974 Metabolism of epidermal tissues, mesophyll cells and bundle sheath strands resolved from mature nutsedge leaves. Arch Biochem Biophys 163: 246-262 9. DOUCE R. J JOYARD 1979 Structure and function of the plastid envelope. Adv Bot Res 7: 1-117 10. DOWNTON WJS, JS HAWKER 1973 Enzymes of starch and sucrose metabblism in Zea mays leaves. Phytochemistry 12: 1551-1556 I 1. FEKETE MAR DE, GH VIEWEG 1973 Synthesis of sucrose in Zea mays leaves.
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Ber Deutsch Bot Ges 86: 227-231 12. GASPERINI C, P PUPILLO 1983 Aldolase isoenzymes of maize leaves. Plant Sci Lett 28: 163-171 13. HATCH MD, T KAGAWA 1973 Enzymes and functional capacities of mesophyll chloroplasts from plants with C4-pathway photosynthesis. Arch Biochem Biophys 159: 842-853 14. HATCH MD, CB OSMOND 1976 Compartmentation and transport in C4 photosynthesis. In CR Stocking, U Heber, eds, Encyclopedia of Plant Physiology (New Series), Vol 3, Transport in Plants. III. Intracellular Interactions and Transport Processes. Springer-Verlag, Heidelberg, pp 144-184 15. HEBER U, HW HELDT 1981 The chloroplast envelope: structure, function and role in leaf metabolism. Annu Rev Plant Physiol 32: 139-168 16. HERBERT M, C BURKHARD, C SCHNARRENBERGER 1979 A survey for isoenzymes of glucosephosphate isomerase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in C3, C4 and Crassulacean-acid-metabolism plants, and green algae. Planta 145: 95-104 17. KRUGER I, C SCHNARRENBERGER 1983 Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves. Eur J Biochem 136: 101-106 18. KRUGER I, C SCHNARRENBERGER 1985 Development of cytosol and chloroplast aldolases during germination of spinach seeds. Planta 164: 109-114 19. LEBHERZ HG, OJ BATES, RA BRADSHAW 1984 cellular fructose-P2 aldolase has a derivatized (blocked) NH2 terminus. J Biol Chem 259: 1132-1135 20. LEBHERZ HG, MM LEADBETTER, RA BRADSHAW 1984 Isolation and characterization of the cytosolic and chloroplast form of spinach leaf fructose diphosphate aldolase. J Biol Chem 259: 1011-1017 2 1. MBAKU SB, GJ FRITZ, G BowEs 1978 Photosynthetic carbohydrate metabolism in isolated leaf cells of Digitaria pentzii. Plant Physiol 62: 510-515 22. MURPHY DJ, DA WALKER 1981 Aldolase from wheat leaves-its properties and subcellular distribution. FEBS Lett 134: 163-166 23. SCHNARRENBERGER C, M HERBERT, I KRUGER 1983 Intracellular compartmentation of isozymes of sugar phosphate metabolism in green leaves. In MC Rattazzi, JG Scandalios, GS Whitt, eds, Isozymes, Vol 8. Liss, New York, pp 23-51 24. SLACK CR, MD HATCH, DJ GOODSCHILD 1969 Distribution of enzymes in mesophyll and parenchyma sheath chloroplasts of maize leaves in relation to the C4-dicarboxylic acid pathway of photosynthesis. Biochem J 1 14: 489498 25. VIEWEG GH, MAR DE FEKETE 1973 Regulation des Stoffwechsels der Starke in Blattern von Zea mays. Ber Deutsch Bot Ges 86: 233-239 26. WALKER DA 1976 Plastids and intracellular transport. In CR Stocking, U Heber, eds, Encyclopedia of Plant Physiology (New Series), Vol 3, Transport in Plants. III. Intracellular Interactions and Transport Processes. SpringerVerlag, Heidelberg, pp 85-136 27. WEEDEN NF, LD GOTTLIEB 1980 The genetics of chloroplast enzymes. J Hered 71: 392-396 28. WHITTINGHAM CP, AJ KEYS, IF BIRD 1979 The enzymology of sucrose synthesis in leaves. In M Gibbs, E Latzko, eds, Encyclopedia of Plant Physiology (New Series), Vol 6, Photosynthesis. II. Photosynthetic Carbon Metabolism and Related Processes. Springer-Verlag, Heidelberg, pp 313326 29. Wu R, E RACKER 1959 Regulatory mechanisms in carbohydrate metabolism. J Biol Chem 234: 1029-1035 30. ZELITCH I 1971 Photosynthesis, Photorespiration and Plant Productivity. Academic Press, New York
