Russian Journal of Plant Physiology, Vol. 49, No. 2, 2002, pp. 211–215. From Fiziologiya Rastenii, Vol. 49, No. 2, 2002, pp. 238–242. Original English Text Copyright © 2002 by Salahas, Cormas, Zervoudakis.
Cold Inactivation of Phosphoenolpyruvate Carboxylase and Pyruvate Orthophosphate Dikinase from the C4 Perennial Plant Atriplex halimus* G. Salahas*, E. Cormas**, and G. Zervoudakis** *Department of Greenhouses and Floriculture, Technological Institute, Mesologgi, Greece **Department of Biology, Laboratory of Plant Physiology, University of Patras, Patras, Greece; fax: 30(631)-58207; e-mail:
[email protected] Received March 15, 2001
Abstract—Phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) cold inactivation was studied in leaf extracts from Atriplex halimus L. Both enzyme activities gradually reduced as the temperature and the total soluble protein decreased. Mg2+ at a concentration of 10 mM stabilized PEPC and PPDK activities against cold inactivation. At low Mg2+ concentration (4 mM), PEPC was strongly protected by phosphoenolpyruvate, glucose-6-phosphate, and, partially, by L-malate, while PPDK was protected by PEP, but not by its substrate, pyruvate. High concentrations of compatible solutes (glycerol, betaine, proline, sorbitol and trehalose) proved to be good protectants for both enzyme activities against cold inactivation. When illuminated leaves were exposed to low temperature, PPDK was partially inactivated, while the activity of PEPC was not altered. Key words: Atriplex halimus - cold inactivation - in vitro stabilization - phosphoenolpyruvate carboxylase pyruvate orthophosphate dikinase
INTRODUCTION Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and pyruvate orthophosphate dikinase (PPDK; EC 2.7.9.1) located in the cytosol and chloroplasts of the C4 mesophyll cells, respectively, are oligomeric enzymes of the C4-photosynthetic pathway [1, 2]. Both enzymes from C4 plant leaf extracts have been reported to be unstable under dilution and/or cold lability [3–5]. The cold lability of PEPC and PPDK has been attributed to the dissociation of the tetrameric enzymic form to dimers or monomers at low temperatures [3, 6]. It has also been found that pH, protein concentration, and a number of compatible solutes modify the cold lability of both enzymes [4, 7–9]. The PPDK cold lability varies widely between C4 plant species [10] and cultivars within species [11, 12]. Recently, a wide variation of PEPC cold inactivation among various C4 plants has been observed [13]. It was also shown that PEPC from two C4 species of Panicum differ in sensitivity to cold inactivation [14]. Moreover, cold treatment of leaves from C4 plants causes a loss of PPDK [7, 10] but not of PEPC activity [15]. Because of the similarity between both enzymes extracted from different plants in terms of molecular mass, oligomericity and inactivation due to dilution and/or cold treatment, we tried to investigate the cold * This article was submitted by the authors in English.
inactivation of PEPC and PPDK from the same C4 plant and enzyme protection by some compatible solutes. MATERIALS AND METHODS Plant material. Atriplex halimus L. is a C4 plant growing naturally at the area of Patras. Mature leaves were collected under full sunlight, placed into plastic bags and taken immediately to the laboratory. Extraction and desalting of extracts. Leaves (1 g) floating on water were illuminated (1 mmol/(m2 s)) at room temperature for 45 min in order to fully activate PEPC and PPDK and then ground in a mortar with purified sea sand and 4 ml of extraction medium containing 100 mM Tris–HCl, pH 7.8, 10 mM MgCl2, 1 mM EDTA, 5 mM 1,4-dithio-DL-threitol (DTT), 2 mM KH2PO4 and 2% (w/v) polyvinylpyrrolidone (PVP) supplemented with a small amount (100 mg) of insoluble PVP. The extract was centrifuged at 8000 g for 5 min, and the clear supernatant was desalted using a 20 × 1 cm column of Sephadex G-25 (Sigma, United States) which had been equilibrated with 50 mM Tris– HCl, pH 7.5, containing 0.1 mM EDTA, 4 mM MgCl2 and 5 mM DTT. The effluent volume, used as desalted extract, was double that layered on the column. All steps were carried out at room temperature. PEPC activity was assayed in 3-ml final volume of a buffer containing 100 mM Tris–HCl, pH 8.0, 5 mM NaHCO3, 10 mM MgCl2, 4.5 units of malate dehydro-
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1.8 1.6
100 PEPC
24°ë 0°ë
0.8 0.6 0.4 0.2 0
PEPC
80 PPDK
1.4 1.2 1.0
PPDK 0.5 1.0 1.5 2.0 Dilution, mg protein/ml
24°ë 0°ë 2.5
Residual activity, %
Enzyme activity, units/mg protein
2.0
60 40 20
0
10 20 Temperature, °ë
30
Fig. 1. PEPC and PPDK activities of desalted extracts after 45-min incubation at 0 or 24°C, at various dilutions made with desalting buffer. For both PEPC and PPDK, the total protein concentration was the same.
Fig. 2. Inactivation of PEPC and PPDK desalted extracts (diluted 1 : 4) after 45-min incubation at the indicated temperature prior to assay at 30°C. Total protein was 0.6 mg/ml.
genase (pig heart enzyme, Sigma), 14 mM NADH, and 4 mM phosphoenolpyruvate (PEP), at 30°C. The reaction was initiated by the addition of the enzyme, and its rate was measured by monitoring NADH utilization at 340 nm. One unit of PEPC activity corresponds to 1 µmol of PEP converted per min at 30°C.
Incubation of desalted extracts is specified in figures and tables.
PPDK activity was assayed at 30°C in a 1-ml final volume of a buffer containing 100 mM Tris–HCl, pH 8.0, 10 mM MgCl2, 5 mM NaHCO3, 0.1 mM EDTA, 1.25 mM pyruvate, 1.25 mM ATP, 2.5 mM KH2PO4, 5 mM DTT, 2 units of malate dehydrogenase (MDH), 0.2 mM NADH, 6 mM glucose-6-phosphate, and 0.3 units of purified PEPC [16]. The reaction rate was measured by monitoring NADH utilization at 340 nm (NADH oxidation through the PEPC/MDH coupled reactions). The PEPC, one of the coupling enzymes, was purified from maize leaves as described by Hatch [7] with minor modifications (specific activity about 25 units per mg protein). It was devoid of PPDK activity. One unit of PPDK activity corresponds to 1 µmol of pyruvate converted per min at 30°C. Protein content in the extracts was determined by the Bradford method [17] using BSA as the protein standard. Data in figures and tables represent mean values from four independent experiments with a standard error of less than 10% of the mean. Incubation of whole leaves. For in vivo cold inactivation, leaves floating on water were illuminated (1 mmol/(m2 s)) for 45 min at 24°C and then exposed to 2°C for 90 min. At intervals of 30 min, 0.5 g leaves were extracted at room temperature with 2 ml of the extraction buffer. Extracts were centrifuged at 5000 g for 60 s, desalted, and assayed immediately (at 30°C) for the activity of PEPC and PPDK.
RESULTS Dilution experiments. As Fig. 1 shows, the cold inactivation of PEPC and PPDK diminishes as the total soluble protein concentration of the desalted extract (diluted with desalting buffer) increases, i.e., the enzymic activities of both enzymes are more stable at lower dilutions of the initial enzymic extract. PPDK, as compared to PEPC, shows a slightly increased cold inactivation in all dilutions examined. At low protein concentration (0.6 mg/ml), the activity of PPDK and PEPC decreased only by 18 and 12%, respectively, following a 45-min incubation at 24°C, whereas it decreased by 78 and 70% at 0°C, in comparison with the initial activities immediately after the dilution (zero time). At a high protein concentration (2.4 mg/ml), the activities of PPDK and PEPC were stable at 24°C, but decreased by 29 and 12%, respectively, at 0°C. The initial activities of PPDK and PEPC were 0.108 and 1.78 units/mg protein and remained unchanged at all dilutions at zero time. In order to evaluate the temperature-dependent inactivation of PEPC and PPDK, the desalted enzyme extracts (diluted 1 : 4) were incubated between 0 and 30°C for 45 min, and the percentage of residual activity was measured. As shown in Fig. 2, a gradual increase in the PEPC and PPDK inactivation was observed as the temperature was lowered from 30 to 0°C. The influence of various substances on PEPC and PPDK cold inactivation was also examined. As shown in Table 1, PEPC activity was largely protected from cold inactivation when 10 mM MgCl2 was included in
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COLD INACTIVATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE Table 1. PEPC and PPDK activities after 45-min incubation of desalted extract (1 : 4) at 24 and 0°C in the presence of various substances
Substance
PEPC
PPDK
24°C
0°C
24°C
0°C
No addition
88
30
82
22
MgCl2, 10 mM
99
86
100
66
100
92
99
77
Pyruvate, 5 mM
83
27
78
24
L-malate, 6 mM
100
59
76
18
Glucose-6-phosphate, 10 mM
100
95
80
17
PEP, 4 mM
Note: Total protein content was 0.6 mg/ml.
the incubation mixture. The enzyme substrate PEP (4 mM) and the allosteric activator and/or stabilizer glucose-6-phosphate (10 mM) provided further protection. The allosteric PEPC inhibitor, L-malate (6 mM), protected the enzyme activity to a lower extent. MgCl2 (10 mM) partially protected PPDK activity against cold inactivation while pyruvate (5 mM) had no effect. However, the PPDK product PEP (4 mM), protected sufficiently the enzyme activity against cold inactivation.
100
4
80
3
2 1
20 10
20 30 40 Incubation time, min
PPDK protection
1.40 0.50 0.18 0.34 0.80
1.52 0.48 0.23 0.40 0.73
Note: Desalted extracts diluted 1 : 4 were incubated as shown in Fig. 3.
sorbitol, trehalose and glycerol, and the concentrations that provide full protection of PEPC activity were determined (Table 2). The activity of PPDK was fully protected against cold inactivation by similar cosolute concentrations. In vivo cold inactivation. The extractable PPDK activity from detached illuminated leaves that were exposed to 2°C for 90 min declined by more than 30% (Fig. 4). In contrast, PEPC extractable activity did not show any substantial decline under the same conditions, indicating that this enzyme cannot be cold labile in vivo. DISCUSSION The in vitro properties of PEPC and PPDK are partly artifact of dilution, because the high protein concentration in the plant cell [14] favors the lower cold sensitivity of enzyme aggregation [3, 6]. Our results are in accordance with previous findings, which proposed that enzyme transformation from tetramer to dimer or monomer is responsible for enzyme inactivation by
50
Fig. 3. Cold inactivation of PEPC desalted extracts (diluted 1 : 4) for 45-min at 0°C in the presence of betaine at concentrations of (1) 0, (2) 0.05, (3) 0.1, and (4) 0.18 M. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
PEPC protection
PEPC
100
60
0
Concentration (M) for
Glycerol Proline Betain Sorbitol Trehalose
Residual activity, %
Residual activity, %
Stabilization by cosolutes. In a series of stabilization experiments, we examined the effect of some cosolutes on PEPC and PPDK cold inactivation. The betaine concentration that provides maximum protection against PEPC cold inactivation was about 180 mM (Fig. 3). A similar protective effect was also exhibited by proline,
40
Table 2. Cosolute concentrations necessary for full protection of PEPC and PPDK activities against cold inactivation Cosolute
Residual activity, %
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80 60
PPDK
40 20 0
20
40 60 80 Incubation time, min
100
Fig. 4. Effects of leaf temperature lowered from 24 to 2°C on the PEPC and PPDK extractable activities. Leaves were detached and illuminated at 24°C to activate both enzymes and then exposed to 2°C. No. 2
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dilution and/or cold inactivation [4, 18, 19]. There have been many reports regarding the inactivation of various enzymes due to dilution-mediated depolymerization from oligomers to monomers [19–22]. The equilibrium between oligomeric forms of PEPC and PPDK with different properties may explain the protection against cold inactivation afforded by high protein concentration as was shown here and reported by other investigators [4, 14, 19]. It has also been proposed that the enzyme instability, due to dilution, is independent of and additive to that caused by cold temperatures, and any treatment that protects against cold should also protect against dilution and vice versa [14]. Cold temperature decreases the enzymic integrity by weakening the hydrophobic intramolecular interactions and/or changing the pK values of ionizable groups leading to enzyme inactivation [4, 14]. Krall and Edwards [14] by using the chaotropic ion SCN– and deuterium oxide (D2O) that destabilize and stabilize hydrophobic bonds, respectively, proved that these bonds are involved in the integrity of the enzymatically active PEPC molecule derived from Panicum. According to our results, PPDK and PEPC from A. halimus are both cold inactivated, the effect being concentration-dependent, but some compatible solutes at high concentrations protect both enzymes against cold inactivation. It was also shown that trehalose, a nonreducing disaccharide found at high concentrations in anhydrobiotic organisms [23] and in resurrection plants [24], protected PEPC and PPDK against cold inactivation. The stabilization of oligomeric proteins by compatible solutes has been attributed to a mechanism outlined by the exclusion volume theory [25, 26]. According to this theory, the compatible solutes are strongly hydrated in aqueous solutions. Since interaction between proteins and compatible solutes is thermodynamically unfavourable, the proteins are restricted to a certain part of the total volume. The physiological significance of PEPC and PPDK cold lability is still uncertain and remains under investigation, since there is evidence, in coincidence with our results, that PEPC is not cold-inactivated in vivo [15, 27] probably because of the differences between the in vitro and the in vivo microenvironments of the enzyme [14]. In contrast, there is evidence that PPDK from the leaves of the C4 plants Digitaria sanguinalis and Sorghum is in vivo cold inactivated [10, 28], which is in accordance to our results. In previous studies, extracted PPDK activities from different C4 plants were just equal or lower than the observed rates of photosynthesis suggesting a rate-limiting role for PPDK [12]. ACKNOWLEDGMENT This work was partially supported by the Greek General Secretariat of Research and Technology, project 91 ED412.
REFERENCES 1. Edwards, G.E., Nakamoto, H., Burnell, J.N., and Hatch, M.D., Pyruvate Pi Dikinase and NADP-Malate Dehydrogenase in C4 Photosynthesis: Properties and Mechanism of Light/Dark Regulation, Annu. Rev. Plant Physiol., 1985, vol. 36, pp. 255–286. 2. Andreo, C.S., González, D.H., and Iglesias, A.A., Higher Plant Phosphoenolpyruvate Carboxylase. Structure and Regulation, FEBS Lett., 1987, vol. 213, pp. 1–8. 3. Shirahashi, K., Hayakawa, S., and Sugiyama, T., Cold Lability of Pyruvate Orthophosphate Dikinase in the Maize Leaf, Plant Physiol., 1978, vol. 62, pp. 826–830. 4. Angelopoulos, K. and Gavalas, N.A., Reversible Cold Inactivation of C4-Phosphoenolpyruvate Carboxylase: Factors Affecting Reactivation and Stability, J. Plant Physiol., 1988, vol. 132, pp. 714–719. 5. Salahas, G., Manetas, Y., and Gavalas, N.A., Effects of Glycerol on the in vitro Stability and Regulatory Activation/Inactivation of Pyruvate Orthophosphate Dikinase of Zea mays L., Photosynth. Res., 1990, vol. 26, pp. 9–17. 6. Willeford, K.O. and Wedding, R.T., Oligomerization and Regulation of Higher Plant Phosphoenolpyruvate Carboxylase, Plant Physiol., 1992, vol. 99, pp. 755–758. 7. Hatch, M.D., Regulation of C4 Photosynthesis: Factors Affecting Cold Mediated Inactivation and Reactivation of Pyruvate Pi Dikinase, Aust. J. Plant Physiol., 1979, vol. 6, pp. 607–619. 8. Walker, G.H., Ku, M.S.B., and Edwards, G.E., Catalytic Activity of Maize Leaf Phosphoenolpyruvate Carboxylase in Relation to Oligomerization, Plant Physiol., 1986, vol. 80, pp. 848–855. 9. Krall, J.P., Edwards, G.E. and Andreo, C.A., Protection of Pyruvate Pi Dikinase from Maize against Cold Lability by Compatible Solutes, Plant Physiol., 1989, vol. 89, pp. 280–285. 10. Sugiyama, T., Schmitt, M.R., Ku, S.B., and Edwards, G.E., Differences in Cold Lability of Pyruvate Pi Dikinase among C4 Species, Plant Cell Physiol., 1979, vol. 20, pp. 965–971. 11. Sugiyama, T. and Boku, K., Differing Sensitivity of Pyruvate Orthophosphate Dikinase to Low Temperature in Maize Cultivars, Plant Cell Physiol., 1976, vol. 17, pp. 851–854. 12. Simon, J.-P. and Hatch, M.D., Temperature Effects on the Activation and Inactivation of Pyruvate Pi Dikinase in Two Populations of the C4 Weed Echinochloa crusgalli (Barnyard Grass) from Sites of Contrasting Climates, Aust. J. Plant Physiol., 1994, vol. 21, pp. 463–473. 13. Zervoudakis, G., Angelopoulos, K., Salahas, G., and Georgiou, C.D., Differences in Cold Inactivation of Phosphoenolpyruvate Carboxylase among C4 Species: The Effect of pH and of Enzyme Concentration, Photosynthetica, 1998, vol. 35, pp. 169–175. 14. Krall, J.P. and Edwards, G.E., PEP carboxylases from Two C4 species of Panicum with Markedly Different Susceptibilities to Cold Inactivation, Plant Cell Physiol., 1993, vol. 34, pp. 1–11. 15. Samaras, Y., Manetas, Y., and Gavalas, N.A., Effects of Temperature and Photosynthetic Inhibitors on Light Activation of C4-Phosphoenolpyruvate Carboxylase, Photosynth. Res., 1988, vol. 16, pp. 233–242.
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COLD INACTIVATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE 16. Salahas, G., Manetas, Y., and Gavalas, N.A., Assaying for Pyruvate Orthophosphate Dikinase Activity: Necessary Precautions with Phosphoenolpyruvate Carboxylase as Coupling Enzyme, Photosynth. Res., 1990, vol. 24, pp. 183–188. 17. Bradford, M.M., A Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem., 1976, vol. 72, pp. 248–254. 18. Shi, J.-N., Wu, M.-X., and Zha, J.-J., Studies on Plant Phosphoenolpyruvate Carboxylase: 5. A Reversible Cold Inactivation of Sorghum Leaf Carboxylase, Acta Phytophysiol. Sinica, 1981, vol. 7, pp. 317–326. 19. Stamatakis, K., Gavalas, N.A., and Manetas, Y., Organic Cosolutes Increase the Catalytic Efficiency of Phosphoenolpyruvate Carboxylase from Cynodon dactylon (L.) Pers., Apparently through Self-Association of the Enzymic Protein, Aust. J. Plant Physiol., 1988, vol. 15, pp. 621–631. 20. Aaronsen, R.P. and Frieden, C., Rabbit Muscle Phosphofructokinase: Studies on the Polymerization, J. Biol. Chem., 1972, vol. 247, pp. 7502–7509. 21. Bock, P.E. and Frieden, C., Another Look on the Cold Lability of Enzymes, Trends Biochem. Sci., 1978, vol. 4, pp. 100–103. 22. Khew-Goodal, Y.S., Johannssen, W., Attwood, P.V., Wallace, J.C., and Keech, D.B., Studies on Dilution Inactiva-
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Vol. 49
23. 24.
25.
26.
27.
28.
215
tion of Sheep Liver Pyruvate Carboxylase, Arch. Biochem. Biophys., 1991, vol. 284, pp. 98–105. Crowe, J.H. and Crowe, L.M., Preservation of Membranes in Anhydrobiotic Organisms: the Role of Trehalose, Science, 1984, vol. 223, pp. 701–703. Bianchi, G., Gamba, A., Limiroli, R., Pozzi, N., Elster, R., Salamini, F., and Bartels, D., The Unusual Sugar Composition in Leaves of the Resurrection Plant Myrothamnus flabellifolia, Physiol. Plant., 1993, vol. 87, pp. 223–226. Gekko, K. and Timasheff, S.N., Mechanism of Protein Stabilization by Glycerol: Preferential Hydration in Glycerol Water Mixtures, Biochemistry, 1981, vol. 20, pp. 4667–4676. Selinioti, E., Nikolopoulos, D., and Manetas, Y., Organic Cosolutes as Stabilizers of Phosphoenolpyruvate Carboxylase in Storage: an Interpretation of Their Action, Aust. J. Plant Physiol., 1987, vol. 14, pp. 203–210. Petropoulou, Y., Manetas, Y., and Gavalas, N.A., Intact Mesophyll Protoplasts from Zea mays as a Source of Phosphoenolpyruvate Carboxylase Unaffected by Extraction: Advantages and Limitations, Physiol. Plant., 1990, vol. 80, pp. 605–611. Taylor, A.O., Slack, C.R. and Mc Pherson, H.G., Plants under Climatic Stress: 6. Chilling and Light Effects on Photosynthetic Enzymes of Sorghum and Maize, Plant Physiol., 1974, vol. 54, pp. 696–701.
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