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Isoenzymes of phosphogluconate dehydrogenase (decarboxylating) from suspension-cultured Catharanthus roseus cells
Phytochemistry,Vol. 33, No. 6, pp. 1307-1311, 1993 Printedin Great Britain.
0
0031 9422/93 $6.00 + 0.00 1993 PergamonPress Ltd
ISOENZYMES OF PHOSPHOGLUCONATE DEHYDROGENASE (DECARBOXYLATING) FROM SUSPENSION-CULTURED CATHARANTHUS ROSEUS CELLS* SARAHMI ISHIDA~
and
HIROSHI ASHIHARA~
Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1, Otsuka, Bunkyo-City, Tokyo 112,Japan (Received 5 January 1993)
Key Word Index-Cutharanthus roseus; Apocynaceae; Madagascar periwinkle; cell culture; 6-phosphogluconate dehydrogenase; pentose phosphate pathway; carbohydrate metabolism.
Abstract-Two isoenzymes of phosphogluconate dehydrogenase (PGD; EC 1.1.1.44) were found in suspensioncultured Catharanrhus roseus cells. They are located in the cytosol and the plastids (amyloplasts). These two isoenzymes were separated by fractionation on an anion-exchange column by HPLC. The two isoenzymes had similar kinetic properties and the same effecters. The K, values for 6-phosphogluconate were 15 and 18 PM and K, values for NADP+ were 8 and 11 PM for the cytosolic and plastid isoenzymes, respectively. NADPH was a potent inhibitor of both isoenzymes. ATP and 5-phosphoribosyl-1-pyrophosphate also inhibited the activities of these isoenzymes. The maximum level of PGD was observed in cells at the lag phase of growth. The activity of cytosolic PGD relative to that of the plastid PGD was also high in these cells. In contrast, the activity of the plastid isoenzyme of PGD was ca twofold higher than that of the cytosolic isoenzyme in the cells at the early stationary phase of growth. The role and regulation of these isoenzymes of PGD are discussed.
INTRODUCTION
RESULTS AND DISCUSSION
In our research on the regulatory mechanisms involved in the catabolism of sugars in suspension-cultured plant cells, we have already investigated the levels and properties of key enzymes [l-4] and we have suggested a possible mechanism for regulation of glycolysis [S]. The oxidative pentose phosphate (PP) pathway represents an alternative route for the catabolism of sugars in plants. The major role of this pathway seems to be the generation of NADPH for use in biosynthetic reactions [6] and also the provision of building blocks for biosynthesis, namely, ribose-5-phosphate for the biosynthesis of nucleotides [7] and erythrose&phosphate for the biosynthesis of aromatic compounds [8]. In the present study, we investigated the subcellular distribution and properties of the isoenzymes of phosphogluconate dehydrogenase [PGD, 6-phospho-D-gluconate: NADP+ 2-oxidoreductase (decarboxylating); EC 1.1.1.44], the second enzyme in the oxidative branch of the PP pathway, in suspensioncultured Catharantkus roseus cells.
PGD is an important enzyme in the PP pathway, because it catalyses the formation of NADPH, which provides essential reducing power for biosynthetic reactions. Compared with the numerous studies of this enzyme in micro-organisms and animals (e.g. [9]), relatively few reports have been published about this enzyme from plant sources [lo]. Recent studies have suggested the presence of duplicate sets of isoenzymes that are involved in the catabolism of sugars, including PGD, in higher plants (for review, see [ 111). Activity staining of a native gel after PAGE of crude extracts of C. roseus cells revealed two bands of active PGD (Fig. 1). The slowly and the rapidly migrating isoenzymes of PGD were designated PGD, and PGD,, respectively. These two isoenzymes were separated by HPLC on an anion-exchange column of Shodex IEC QA824. PGD, and PGDl eluted at ca 0.1 and 0.2 M KCl, respectively. The subcellular distribution of the isoenzymes of PGD was investigated in protoplasts prepared from cultured C. roseus cells. Most of the activity of PGD from burst protoplasts was recovered in the fraction that was pelleted by centrifugation at 1000 g for 1 min and in the supernatant after centrifugation at 12000 g for 20 min. More than 90% of the activities of ADPG pyrophosphorylase and alcohol dehydrogenase, marker enzymes of the plastids (amyloplasts) [ 121 and the cytosol [13], respectively, were recovered in these two fractions (Table 1). The PGD in these two fractions was further
*Part 42 in the series, ‘Metabolic Regulation in Plant Cell Culture’. TPresent address: Department of Botany, Faculty of Science, University of Tokyo, Hongo, Bunkyo-City, Tokyo 113, Japan. fAuthor to whom correspondence should be addressed.
1307
S. ISHIDA and H. ASHIHARA
1308
-25 Fraction number
0
25
~/[NAD~*I
Fig. 1. Elutlon profiles of isoenzymes of PGD from an anion exchange column (Shodex IEC QA-824) after HPLC. Insets show the distribution of activities of PGD on 7.5% polyacrylamide gels. (1) PGD,: (2) PGD,: (1) +(2), mixture of the two.
50 (mM_‘)
Fig. 2. Inhibition of NADPH of the activity of PGD, from C’atharanthus roseus cells. The concentrations of NADPH were 0, none; 0,2.5 PM; A. 50 PM; C, 100 PM. A similar result was obtained with the other lsoenzyme (PGD,).
Table 1. Subcellular distribution of phosphogluconate dehydrogenase (PGD), alcohol dehydrogenase (ADH) and ADP-glucose pyrophosphorylase (ADPGPP) in suspension-cultured Catharunthus roseus cells Plastid
Fraction PGD Total activity (nkat) Specific activity (nkat mg-’ ADH Total activity (nkat) Specific activity (nkat mg-’ ADPGPP Total activity (nkat) Specific activity (nkat mg-’ The values cultures.
were obtained
protein)
protein)
protein)
2.5 (34%) 4.8 6.3 (5%) 12.2 1.2 (92%) 2.3
from cells harvested
analysed by PAGE on native gels. Only a single band of activity corresponding to PGD, was detected in the cytosolic fraction, whereas two bands, a strong and a faint band, were found in the plastid fraction. The major band of activity corresponded to PGD,. The staining of the faint band was found to be ca lOO-fold weaker than that of the main band, by densitometric scanning. These results suggest that PGD, and PGD, are enzymes located in the cytosol and the plastids (amyloplasts), respectively. Cytosolic and plastidic isoenzymes of PGD have also been reported in pea roots [ 14,151, castor bean endosperm [ 161, spinach leaves [ 173 and the host fraction of soybean nodules [18]. Macherel et al. [19] also reported that extracts of amyloplasts obtained from cultured sycamore cells exhibited substantial PGD activity. Kinetic properties of the isoenzymes were investigated using partially purified preparations of PGD, and PGD,. Both isoenzymes showed hyperbolic kinetics for their two substrates, 6-phosphogluconate (6PG) and NADP+. The pi, values of PGD, for 6PG and NADP+ were 15 and 8 PM, respectively, and those of PGD, were 18 (6PG) and
Cytosol
4.8 (66%) 2.5 109.3 (95%) 57.0 0.10 (8%) 0.05 from five-day-old
I1 PM (NADP+). Similar K, values for 6PG were obtained for PGD from black gram (7 PM) [20], carrot (6 PM) [20] and pea roots (1 I and 23 PM) [14], but these values are lower than those obtained for PGD from soybean nodules (98 and 109 PM) [ 181. A high affinity for NADP+ had been reported in several PGDs obtained from higher plants, including soybean nodules [14.16-l 8. 20, 213. The activity of both isoenzymes of PGD from C. roseus cells was inhibited by NADPH, ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP). NADPH was the most potent inhibitor of the enzyme. The inhibition was competitive with respect to NADP+ (Fig. 2) and K, values were 19 (PGD,) and 25 PM (PGD,). NADPH is also a strong inhibitor of glucose-6-phosphate dehydrogenase from higher plants [22]. Thus, the ratio of NADPH : NADP ’ appears to be most important in the regulation of the oxidative route of the PP pathway in C. roseus cells, as has been proposed in other plants [lo, 231. The activities of PGD, and PGD, were inhibited by ATP. This inhibition appeared to be competitive with respect to 6PG with Ki values of 1.6 and 1.8 mM for
Phosphogluconate
dehydrogenase isoenzymes
PGD, and PGD,, respectively. Since the estimated concentration of ATP in the cytoplasm in cultured C. roseus cells is 3.2 mM [S], inhibition by ATP would seem to occur in viva. PRPP was also a competitive inhibitor of both PGD, and PGD,, and Ki values were 0.20 and 0.18 mM. The inhibition of PRPP, the ribose phosphate donor for the biosynthesis of nucleotides, on the activity
Age of culture
(days)
Fig 3. Changes in the activity of PGD (0) during growth of Catharanthus roseus cells in batch suspension culture. Changes in activity of glucose-6-phosphate dehydrogenasc (0) and fresh weight are also shown. The average values and s.d. were obtained from more than four samples.
1309
of PGD may function as a form of feedback control since the PP pathway plays a role in supplying building blocks for the biosynthesis of nucleotides and nucleic acids. However, in contrast to those of ATP, cellular levels of PRPP in C. roseus cells are extremely low, varying between 0.41 and 2.19 nmolg- ’ fr. wt during culture [24]. Thus, the estimated concentration of PRPP in the cytoplasm is 8-40 PM, when it is calculated on the assumption that the cytoplasmic volume is 5% of the cell volume [25]. These estimated cytoplasmic concentrations of PRPP are five- to 25fold lower than the Ki values of the enzyme for PRPP. Inhibition by PRPP of the activity of PGD has been reported in black gram [20] and Breuibacterium~auum [26] with the relatively low Ki values of 10 and 32 PM, respectively. In the next experiments, as to analyse the coarse control of PGD, we examined fluctuations in the maximum catalytic activity of PGD during growth of C. rosew cells in batch suspension culture (Fig. 3). For comparison, the activity of glucosed-phosphate dehydrogenase [EC 1.1.1.493, another dehydrogenase in the PP pathway, was also monitored (Fig. 3). The activities of both enzymes, expressed as nkat g-r fr. wt, incrased rapidly just after the inoculation of lo-day-old cells from a culture at stationary phase into fresh medium. The levels were maintained during the lag phase of cell growth (days O-4) and then decreased during the exponential phase of growth (days 47). The levels were almost constant at the stationary phase (days 7-12). Figure 4 shows the relative activities of the two isoenzymes of PGD that were obtained from activity staining of nondenaturing gels after PAGE. The relative activity of the
(b)
(a)
f
1 1
e/ ‘l,
(_)
l-day-old
(*)
7-day-old
Fig. 4. Distribution of PGD after polyacrylamide gel electrophoresis Results are shown for cells from one-day-old (a) and seven-day-old (b) cultures of Catharanthus roseus cells. (1) PGD, (cytosolic isoenzyme); (2) PGD, (plastid isoenzyme). TD, tracking dye front.
S. ISHIDAand H. ASHIHARA
1310
cytosolic isoenzyme (PGDi) was 1.6 times higher than that of plastid isoenzyme (PGD2) in cells at the lag phase of growth (day 1). These results suggest that the initial increase in the activity of PGD is mainly attributable to an increase in the level of the cytosolic isoenzyme of PGD. This isoenzyme seems to function as a supplier of NADPH as-well as of building blocks for the biosynthesis of nucleotides and aromatic amino acids, which probably occur in the cytosol. In batch suspension cultures of Acer pseudoplatanus, it has been also suggested that the initial or lag phase of growth is characterized by increased activity of the PP pathway relative to glycolysis [27]. By contrast, the relative activity of the plastid isoenzyme (PGD,) was 1.9-fold higher than that of PGD, in the cells at the stationary phase (day 7). It has been suggested that PGD supplies NADPH for the reduction of nitrite in plastids [28, 291. Furthermore, Emes and Fowler [28] demonstrated that the activity of the plastid isoenzyme of PGD increased in pea roots upon treatment with NO;. In similar batch suspension culture of C. roseus, NHf ions in the culture medium are initally taken up and subsequently NO; ions are absorbed by the cells (H. Ashihara, unpublished results). These findings suggest that the increase in the activity of PGD, at the later stages of growth of cells is, at least in part, due to the synthesis of PGD, that is induced by NO; and this isoenzyme acts to supply NADPH for the reduction of nitrite in plastids. EXPERIMENTAL
Cell cultures. Suspension cultures of C. roseus (L.) G. Don (= Vim-a rosea L.), strain B/S, were maintained in slightly modified MurashigeSkoog medium [30] that consisted of MS basal salts mixt. (Flow Laboratories, Irvine, U.K.), 1.2 PM thiamine-HCI, 2.2 pM 2,4-dichlorophenoxyacetic acid and 3% sucrose. Portions (7 ml) of a suspension containing ca 1 g fr. wt of IO-day-old cells were transferred to 43 ml of fresh medium in 300-ml conical flasks. The cultures were incubated at 27” on a horizontal rotary shaker which was operated at 90 strokes per min, 80 mm amplitude, in the dark. Separation of isoenzymes of PGD. Catharanthus roseus cells were homogenized in a glass homogenizer with
50 mM imidazole buffer (pH 7.6) that contained 2 mM MgCI,, 1 mM sodium EDTA and 1% 2-mercaptoethanol. The homogenate was centrifuged at 30000g for 15 min at 2” and the resulting supernatant was treated with (NH&SO,. The proteins that precipitated at 60% satn were collected by centrifugation and dissolved in 2.5 ml of HEPES-NaOH buffer @H 7.2). A portion of the desalted protein fr. was fractionated by HPLC on an anion-exchange Showdex IEC QA-824 column (Showa Denko, Tokyo, Japan). The column was equilibrated with 20mM Tris-HCI buffer (pH 8.2) that contained 0.1 M KCI. The programme for elution was as follows: O-10 min, 0.1 M KCI; 10-50 mitt, a linear increase from 0.1 to 0.5 M KCI; 50-70 min, 0.5 M KCI. The flow rate was 0.9 ml min - ’ and frs of 0.9 ml were collected. Preparation of PGD from plastids and cytosol. Cells in 5day-old cultures were harvested by filtration through
Miracloth (Calbiochem. La Jolla, CA, U.S.A.) and washed with H,O. A portion of washed cells (3 g fr. wt) was incubated in 15 ml of 0.4 M mannitol soln that contained 1.3% cellulase Onozuka R-10 and 0.7% Macerozyme R10 (Yakult Honsha, Tokyo, Japan) for 4 hr at 27”, with gentle agitation by hand at intervals of 30min. The suspension of protoplasts was filtered through a layer of nylon mesh (pore diameter, 62 pm). The filtrate was carefully transferred to 4 round-bottomed plastic centrifuge tubes (12 mm in diameter, 100 mm in length) and centrifuged at 200 g for 3 min. After the medium had been removed with a Pasteur pipette from each tube, a small amount of 0.4 M mannitol soln (1 ml per tube) was carefully added to the pellet, which was suspended by hand-rotating the tube. Further mannitol soln (2 ml per tube) was added and the mixt. was centrifuged at 200 g for 3 min. After the washing procedure had been repeated 3 x , washed protoplasts were suspended in 1 ml tube-’ of 25 mM HEPES-NaOH buffer (pH 7.6) that contained 0.3 M mannitol, 5 mM MgCl,, 5 mM KC1 and 1 mM dithiothreitol. The suspensions were combined and protoplasts were disrupted by centrifugation at 2000g for 10 set through nylon mesh (pore diameter, 10 pm) which had been positioned in an Eppendorf-type centrifugation tube. Fractionation of cellular organelles was performed by centrifugation at 1000 g for 1 min (plastids in the pellet) and then at 12 000 g for 20 min (mitochondria in the pellet); and the supernatant used as the cytosol. For washing, the pellets were resuspended in mannitol soln that contained HEPES buffer, as mentioned above, and centrifuged again under the same gravitational force. The washed particulate matter was suspended in 25 mM HEPES buffer that contained 5 mM MgCl,, 5 mM KC1 and 1 mM dithiothreitol. The suspension was frozen and thawed twice with liquid N, to destroy membrane structures, and centrifuged at 18 000 g for 15 min. The supernatant obtained was used for assays of enzymes. Preparation of enzymes for detection of maximum catalytic activity. The extracts were prepared as described in
our earlier paper [S]. Assays of enzymatic activities. The activities of enzymes were measured by monitoring changes in A at 340 nm at 30” in a double-beam spectrophotometer. The standard reaction mixt. contained the following components in a final vol. of 1 ml. PGD: 50 mM HEPES (pH 7.4), 0.25 mM 6-phosphogluconate, 0.2 mM NADP+, 5 mM MgCl,. G6PD: 50 mM HEPES (pH 7.4), 2 mM glucose 6-phosphate, 0.2mM NADP+, 5 mM MgCI, and 17 nkat PGD (from yeast). ADH: 50 mM HEPES (pH 7.4), 17.5 mM acetoaldehyde, 0.2 mM NADH. ADPGPP: 1 mM ATP, 1 mM glucose l-phosphate and an excess of pyrophosphate assay reagent (no. P 7275, Sigma, St Louis, MO, U.S.A.). Gel electrophoresis. Non-denaturing gels containing 7.5% polyacrylamide were used [31]. Gels were stained for PGD activity in a reaction mixt. that contained 200 mM HEPES buffer (pH 7.4) 1.25 mM 6-phosphogluconate, 1 mM NADP+, 13 mM MgCI,, 83 pgml- l phenazine methosulphate and 500 p g ml- 1 p-nitroblue tetrazolium.
Phosphogluconate dehydrogenase iseenzymes REFERENCES
1. Ashihara, H. and Horikosi, T. (1987) Z. Naturfirsch. 42c, 1215. 2. Ashihara, H., Horikosi, T., Li, X.-N., Sagishima, K. and Yamashita, Y. (1988) J. Plant Physiol. 133, 38. 3. Yamashita, Y. and Ashihara, H. (1988) Z. Naturforsch. 43e, 827. 4. Sagishima, K., Kubota, K. and Ashihara, H. (1989) Ann. Bot. 64, 185. 5. Kubota, K. and Ashihara, H. (1990) Biochim. Biophys. Acta 1036, 138. 6. Ashihara, H., Komamine, A. and Shimokoriyama, M. (1974) Bot. Mag. Tokyo 87, 121. 7. Hirose, F. and Ashihara, H. (1984) Plant Sci. Letters 35, 123. 8. Hrazdina, G. and Jensen, R. A. (1992) A. Reo. Plant Physiol. Molec. Biol. 43, 241. 9. Pontremoli, S., De Flora, A., Grazi, E., Mangiarotti, G., Bonsignore, A., Horecker, B. L. (1960) J. Biol. Chem. 236, 2975.
L. and Turner, J. F. (1987) in The Biochemistry of Plants (Davies, D. D., ed.), Vol. 11,
10. Copeland,
pp. 107-128. Academic Press, San Diego. 11. Schnarrenberger, C., Herbert, M. and Kruger, I. (1983) in Isozymes: Current Topics in Biological and Medical Research (Rattazzi, M. C., ed.), Vol. 8, pp. 23-51. Alan R. Liss, New York. 12. Keeling, P. L. (1991) in Compartment&on of Plant Metabolism in Non-Photosynthetic Tissues (Emes, M. J., ed.), pp. 111-125. Cambridge University Press, Cambridge. 13. Tintemann, H., Wasternack, C., Helbing, D., Glund, K. and Hause, B. B. (1987) Comp. Biochem. Physiol. 88B, 943.
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14. Ashihara, H. and Fowler, M. W. (1979) Int. J. Biothem. 10, 675. 15. Emes, M. J., Ashihara, H. and Fowler, M. W. (1979) Fedn Eur. Biochem. Sots. Letters 105, 370. 16. Simcox, P. D. and Dennis, D. T. (1978) Plant Physiol. 62, 287. 17. Schnarrenberger, C., Oester, A. and Tolbert, N. E. (1973) Arch. Biochem. Biophys. 154, 438. 18. Hong, Z. Q. and Copeland, L. (1992) J. Plant Physiol. 139, 313. 19. Macherel, D., Viale, A. and Akazawa, T. (1986) Plant Physiol. 80, 1041. 20. Ashihara, H. and Komamine, A. (1975) Int. J. Biothem. 6, 667.
21. Al-Quadan, F., Wender, S. H. and Smith, E. C. (1981) Phytochemistry 20, 961. 22. Ashihara, H. and Komamine, A. (1976) Physiol. Plant. 36, 52. 23. Ashihara, H. and Komamine, A. (1974) Z. PjZanzenphysiol. 74, 130. 24. Hirose, F. and Ashihara, H. (1983) Z. Pjlanzenphysiol.
110, 183. 25. Wagner, K. G. and Backer, A. I. (1992) in International Reoiew ofCytology (Joen, K. W. and Friedlender, M., eds), Vol. 134, pp. l-84. Academic Press, San Diego. 26. Sugimoto, S. and Shiio, I. (1987) Agrie. Biol. Chem. 51, 1257. 27. Fowler, M. W. (1971) J. Exp. Bot. 22, 715. 28. Emes, M. J. and Fowler, M. W. (1983) Planta 158,97. 29. Oji, Y., Watanabe, M., Wakiuchi, N. and Okamoto, S. (1985) Planta 165, 85. 30. Linsmaier, E. M. and Skoog, F. (1965) Physiol. Plant. 18 100. 31. Laemmli, U. K. (1970) Nature (London) 227, 680.
0
0031 9422/93 $6.00 + 0.00 1993 PergamonPress Ltd
ISOENZYMES OF PHOSPHOGLUCONATE DEHYDROGENASE (DECARBOXYLATING) FROM SUSPENSION-CULTURED CATHARANTHUS ROSEUS CELLS* SARAHMI ISHIDA~
and
HIROSHI ASHIHARA~
Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1, Otsuka, Bunkyo-City, Tokyo 112,Japan (Received 5 January 1993)
Key Word Index-Cutharanthus roseus; Apocynaceae; Madagascar periwinkle; cell culture; 6-phosphogluconate dehydrogenase; pentose phosphate pathway; carbohydrate metabolism.
Abstract-Two isoenzymes of phosphogluconate dehydrogenase (PGD; EC 1.1.1.44) were found in suspensioncultured Catharanrhus roseus cells. They are located in the cytosol and the plastids (amyloplasts). These two isoenzymes were separated by fractionation on an anion-exchange column by HPLC. The two isoenzymes had similar kinetic properties and the same effecters. The K, values for 6-phosphogluconate were 15 and 18 PM and K, values for NADP+ were 8 and 11 PM for the cytosolic and plastid isoenzymes, respectively. NADPH was a potent inhibitor of both isoenzymes. ATP and 5-phosphoribosyl-1-pyrophosphate also inhibited the activities of these isoenzymes. The maximum level of PGD was observed in cells at the lag phase of growth. The activity of cytosolic PGD relative to that of the plastid PGD was also high in these cells. In contrast, the activity of the plastid isoenzyme of PGD was ca twofold higher than that of the cytosolic isoenzyme in the cells at the early stationary phase of growth. The role and regulation of these isoenzymes of PGD are discussed.
INTRODUCTION
RESULTS AND DISCUSSION
In our research on the regulatory mechanisms involved in the catabolism of sugars in suspension-cultured plant cells, we have already investigated the levels and properties of key enzymes [l-4] and we have suggested a possible mechanism for regulation of glycolysis [S]. The oxidative pentose phosphate (PP) pathway represents an alternative route for the catabolism of sugars in plants. The major role of this pathway seems to be the generation of NADPH for use in biosynthetic reactions [6] and also the provision of building blocks for biosynthesis, namely, ribose-5-phosphate for the biosynthesis of nucleotides [7] and erythrose&phosphate for the biosynthesis of aromatic compounds [8]. In the present study, we investigated the subcellular distribution and properties of the isoenzymes of phosphogluconate dehydrogenase [PGD, 6-phospho-D-gluconate: NADP+ 2-oxidoreductase (decarboxylating); EC 1.1.1.44], the second enzyme in the oxidative branch of the PP pathway, in suspensioncultured Catharantkus roseus cells.
PGD is an important enzyme in the PP pathway, because it catalyses the formation of NADPH, which provides essential reducing power for biosynthetic reactions. Compared with the numerous studies of this enzyme in micro-organisms and animals (e.g. [9]), relatively few reports have been published about this enzyme from plant sources [lo]. Recent studies have suggested the presence of duplicate sets of isoenzymes that are involved in the catabolism of sugars, including PGD, in higher plants (for review, see [ 111). Activity staining of a native gel after PAGE of crude extracts of C. roseus cells revealed two bands of active PGD (Fig. 1). The slowly and the rapidly migrating isoenzymes of PGD were designated PGD, and PGD,, respectively. These two isoenzymes were separated by HPLC on an anion-exchange column of Shodex IEC QA824. PGD, and PGDl eluted at ca 0.1 and 0.2 M KCl, respectively. The subcellular distribution of the isoenzymes of PGD was investigated in protoplasts prepared from cultured C. roseus cells. Most of the activity of PGD from burst protoplasts was recovered in the fraction that was pelleted by centrifugation at 1000 g for 1 min and in the supernatant after centrifugation at 12000 g for 20 min. More than 90% of the activities of ADPG pyrophosphorylase and alcohol dehydrogenase, marker enzymes of the plastids (amyloplasts) [ 121 and the cytosol [13], respectively, were recovered in these two fractions (Table 1). The PGD in these two fractions was further
*Part 42 in the series, ‘Metabolic Regulation in Plant Cell Culture’. TPresent address: Department of Botany, Faculty of Science, University of Tokyo, Hongo, Bunkyo-City, Tokyo 113, Japan. fAuthor to whom correspondence should be addressed.
1307
S. ISHIDA and H. ASHIHARA
1308
-25 Fraction number
0
25
~/[NAD~*I
Fig. 1. Elutlon profiles of isoenzymes of PGD from an anion exchange column (Shodex IEC QA-824) after HPLC. Insets show the distribution of activities of PGD on 7.5% polyacrylamide gels. (1) PGD,: (2) PGD,: (1) +(2), mixture of the two.
50 (mM_‘)
Fig. 2. Inhibition of NADPH of the activity of PGD, from C’atharanthus roseus cells. The concentrations of NADPH were 0, none; 0,2.5 PM; A. 50 PM; C, 100 PM. A similar result was obtained with the other lsoenzyme (PGD,).
Table 1. Subcellular distribution of phosphogluconate dehydrogenase (PGD), alcohol dehydrogenase (ADH) and ADP-glucose pyrophosphorylase (ADPGPP) in suspension-cultured Catharunthus roseus cells Plastid
Fraction PGD Total activity (nkat) Specific activity (nkat mg-’ ADH Total activity (nkat) Specific activity (nkat mg-’ ADPGPP Total activity (nkat) Specific activity (nkat mg-’ The values cultures.
were obtained
protein)
protein)
protein)
2.5 (34%) 4.8 6.3 (5%) 12.2 1.2 (92%) 2.3
from cells harvested
analysed by PAGE on native gels. Only a single band of activity corresponding to PGD, was detected in the cytosolic fraction, whereas two bands, a strong and a faint band, were found in the plastid fraction. The major band of activity corresponded to PGD,. The staining of the faint band was found to be ca lOO-fold weaker than that of the main band, by densitometric scanning. These results suggest that PGD, and PGD, are enzymes located in the cytosol and the plastids (amyloplasts), respectively. Cytosolic and plastidic isoenzymes of PGD have also been reported in pea roots [ 14,151, castor bean endosperm [ 161, spinach leaves [ 173 and the host fraction of soybean nodules [18]. Macherel et al. [19] also reported that extracts of amyloplasts obtained from cultured sycamore cells exhibited substantial PGD activity. Kinetic properties of the isoenzymes were investigated using partially purified preparations of PGD, and PGD,. Both isoenzymes showed hyperbolic kinetics for their two substrates, 6-phosphogluconate (6PG) and NADP+. The pi, values of PGD, for 6PG and NADP+ were 15 and 8 PM, respectively, and those of PGD, were 18 (6PG) and
Cytosol
4.8 (66%) 2.5 109.3 (95%) 57.0 0.10 (8%) 0.05 from five-day-old
I1 PM (NADP+). Similar K, values for 6PG were obtained for PGD from black gram (7 PM) [20], carrot (6 PM) [20] and pea roots (1 I and 23 PM) [14], but these values are lower than those obtained for PGD from soybean nodules (98 and 109 PM) [ 181. A high affinity for NADP+ had been reported in several PGDs obtained from higher plants, including soybean nodules [14.16-l 8. 20, 213. The activity of both isoenzymes of PGD from C. roseus cells was inhibited by NADPH, ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP). NADPH was the most potent inhibitor of the enzyme. The inhibition was competitive with respect to NADP+ (Fig. 2) and K, values were 19 (PGD,) and 25 PM (PGD,). NADPH is also a strong inhibitor of glucose-6-phosphate dehydrogenase from higher plants [22]. Thus, the ratio of NADPH : NADP ’ appears to be most important in the regulation of the oxidative route of the PP pathway in C. roseus cells, as has been proposed in other plants [lo, 231. The activities of PGD, and PGD, were inhibited by ATP. This inhibition appeared to be competitive with respect to 6PG with Ki values of 1.6 and 1.8 mM for
Phosphogluconate
dehydrogenase isoenzymes
PGD, and PGD,, respectively. Since the estimated concentration of ATP in the cytoplasm in cultured C. roseus cells is 3.2 mM [S], inhibition by ATP would seem to occur in viva. PRPP was also a competitive inhibitor of both PGD, and PGD,, and Ki values were 0.20 and 0.18 mM. The inhibition of PRPP, the ribose phosphate donor for the biosynthesis of nucleotides, on the activity
Age of culture
(days)
Fig 3. Changes in the activity of PGD (0) during growth of Catharanthus roseus cells in batch suspension culture. Changes in activity of glucose-6-phosphate dehydrogenasc (0) and fresh weight are also shown. The average values and s.d. were obtained from more than four samples.
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of PGD may function as a form of feedback control since the PP pathway plays a role in supplying building blocks for the biosynthesis of nucleotides and nucleic acids. However, in contrast to those of ATP, cellular levels of PRPP in C. roseus cells are extremely low, varying between 0.41 and 2.19 nmolg- ’ fr. wt during culture [24]. Thus, the estimated concentration of PRPP in the cytoplasm is 8-40 PM, when it is calculated on the assumption that the cytoplasmic volume is 5% of the cell volume [25]. These estimated cytoplasmic concentrations of PRPP are five- to 25fold lower than the Ki values of the enzyme for PRPP. Inhibition by PRPP of the activity of PGD has been reported in black gram [20] and Breuibacterium~auum [26] with the relatively low Ki values of 10 and 32 PM, respectively. In the next experiments, as to analyse the coarse control of PGD, we examined fluctuations in the maximum catalytic activity of PGD during growth of C. rosew cells in batch suspension culture (Fig. 3). For comparison, the activity of glucosed-phosphate dehydrogenase [EC 1.1.1.493, another dehydrogenase in the PP pathway, was also monitored (Fig. 3). The activities of both enzymes, expressed as nkat g-r fr. wt, incrased rapidly just after the inoculation of lo-day-old cells from a culture at stationary phase into fresh medium. The levels were maintained during the lag phase of cell growth (days O-4) and then decreased during the exponential phase of growth (days 47). The levels were almost constant at the stationary phase (days 7-12). Figure 4 shows the relative activities of the two isoenzymes of PGD that were obtained from activity staining of nondenaturing gels after PAGE. The relative activity of the
(b)
(a)
f
1 1
e/ ‘l,
(_)
l-day-old
(*)
7-day-old
Fig. 4. Distribution of PGD after polyacrylamide gel electrophoresis Results are shown for cells from one-day-old (a) and seven-day-old (b) cultures of Catharanthus roseus cells. (1) PGD, (cytosolic isoenzyme); (2) PGD, (plastid isoenzyme). TD, tracking dye front.
S. ISHIDAand H. ASHIHARA
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cytosolic isoenzyme (PGDi) was 1.6 times higher than that of plastid isoenzyme (PGD2) in cells at the lag phase of growth (day 1). These results suggest that the initial increase in the activity of PGD is mainly attributable to an increase in the level of the cytosolic isoenzyme of PGD. This isoenzyme seems to function as a supplier of NADPH as-well as of building blocks for the biosynthesis of nucleotides and aromatic amino acids, which probably occur in the cytosol. In batch suspension cultures of Acer pseudoplatanus, it has been also suggested that the initial or lag phase of growth is characterized by increased activity of the PP pathway relative to glycolysis [27]. By contrast, the relative activity of the plastid isoenzyme (PGD,) was 1.9-fold higher than that of PGD, in the cells at the stationary phase (day 7). It has been suggested that PGD supplies NADPH for the reduction of nitrite in plastids [28, 291. Furthermore, Emes and Fowler [28] demonstrated that the activity of the plastid isoenzyme of PGD increased in pea roots upon treatment with NO;. In similar batch suspension culture of C. roseus, NHf ions in the culture medium are initally taken up and subsequently NO; ions are absorbed by the cells (H. Ashihara, unpublished results). These findings suggest that the increase in the activity of PGD, at the later stages of growth of cells is, at least in part, due to the synthesis of PGD, that is induced by NO; and this isoenzyme acts to supply NADPH for the reduction of nitrite in plastids. EXPERIMENTAL
Cell cultures. Suspension cultures of C. roseus (L.) G. Don (= Vim-a rosea L.), strain B/S, were maintained in slightly modified MurashigeSkoog medium [30] that consisted of MS basal salts mixt. (Flow Laboratories, Irvine, U.K.), 1.2 PM thiamine-HCI, 2.2 pM 2,4-dichlorophenoxyacetic acid and 3% sucrose. Portions (7 ml) of a suspension containing ca 1 g fr. wt of IO-day-old cells were transferred to 43 ml of fresh medium in 300-ml conical flasks. The cultures were incubated at 27” on a horizontal rotary shaker which was operated at 90 strokes per min, 80 mm amplitude, in the dark. Separation of isoenzymes of PGD. Catharanthus roseus cells were homogenized in a glass homogenizer with
50 mM imidazole buffer (pH 7.6) that contained 2 mM MgCI,, 1 mM sodium EDTA and 1% 2-mercaptoethanol. The homogenate was centrifuged at 30000g for 15 min at 2” and the resulting supernatant was treated with (NH&SO,. The proteins that precipitated at 60% satn were collected by centrifugation and dissolved in 2.5 ml of HEPES-NaOH buffer @H 7.2). A portion of the desalted protein fr. was fractionated by HPLC on an anion-exchange Showdex IEC QA-824 column (Showa Denko, Tokyo, Japan). The column was equilibrated with 20mM Tris-HCI buffer (pH 8.2) that contained 0.1 M KCI. The programme for elution was as follows: O-10 min, 0.1 M KCI; 10-50 mitt, a linear increase from 0.1 to 0.5 M KCI; 50-70 min, 0.5 M KCI. The flow rate was 0.9 ml min - ’ and frs of 0.9 ml were collected. Preparation of PGD from plastids and cytosol. Cells in 5day-old cultures were harvested by filtration through
Miracloth (Calbiochem. La Jolla, CA, U.S.A.) and washed with H,O. A portion of washed cells (3 g fr. wt) was incubated in 15 ml of 0.4 M mannitol soln that contained 1.3% cellulase Onozuka R-10 and 0.7% Macerozyme R10 (Yakult Honsha, Tokyo, Japan) for 4 hr at 27”, with gentle agitation by hand at intervals of 30min. The suspension of protoplasts was filtered through a layer of nylon mesh (pore diameter, 62 pm). The filtrate was carefully transferred to 4 round-bottomed plastic centrifuge tubes (12 mm in diameter, 100 mm in length) and centrifuged at 200 g for 3 min. After the medium had been removed with a Pasteur pipette from each tube, a small amount of 0.4 M mannitol soln (1 ml per tube) was carefully added to the pellet, which was suspended by hand-rotating the tube. Further mannitol soln (2 ml per tube) was added and the mixt. was centrifuged at 200 g for 3 min. After the washing procedure had been repeated 3 x , washed protoplasts were suspended in 1 ml tube-’ of 25 mM HEPES-NaOH buffer (pH 7.6) that contained 0.3 M mannitol, 5 mM MgCl,, 5 mM KC1 and 1 mM dithiothreitol. The suspensions were combined and protoplasts were disrupted by centrifugation at 2000g for 10 set through nylon mesh (pore diameter, 10 pm) which had been positioned in an Eppendorf-type centrifugation tube. Fractionation of cellular organelles was performed by centrifugation at 1000 g for 1 min (plastids in the pellet) and then at 12 000 g for 20 min (mitochondria in the pellet); and the supernatant used as the cytosol. For washing, the pellets were resuspended in mannitol soln that contained HEPES buffer, as mentioned above, and centrifuged again under the same gravitational force. The washed particulate matter was suspended in 25 mM HEPES buffer that contained 5 mM MgCl,, 5 mM KC1 and 1 mM dithiothreitol. The suspension was frozen and thawed twice with liquid N, to destroy membrane structures, and centrifuged at 18 000 g for 15 min. The supernatant obtained was used for assays of enzymes. Preparation of enzymes for detection of maximum catalytic activity. The extracts were prepared as described in
our earlier paper [S]. Assays of enzymatic activities. The activities of enzymes were measured by monitoring changes in A at 340 nm at 30” in a double-beam spectrophotometer. The standard reaction mixt. contained the following components in a final vol. of 1 ml. PGD: 50 mM HEPES (pH 7.4), 0.25 mM 6-phosphogluconate, 0.2 mM NADP+, 5 mM MgCl,. G6PD: 50 mM HEPES (pH 7.4), 2 mM glucose 6-phosphate, 0.2mM NADP+, 5 mM MgCI, and 17 nkat PGD (from yeast). ADH: 50 mM HEPES (pH 7.4), 17.5 mM acetoaldehyde, 0.2 mM NADH. ADPGPP: 1 mM ATP, 1 mM glucose l-phosphate and an excess of pyrophosphate assay reagent (no. P 7275, Sigma, St Louis, MO, U.S.A.). Gel electrophoresis. Non-denaturing gels containing 7.5% polyacrylamide were used [31]. Gels were stained for PGD activity in a reaction mixt. that contained 200 mM HEPES buffer (pH 7.4) 1.25 mM 6-phosphogluconate, 1 mM NADP+, 13 mM MgCI,, 83 pgml- l phenazine methosulphate and 500 p g ml- 1 p-nitroblue tetrazolium.
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