Control of ornithine decarboxylase activity in jute seeds by antizyme

J. Biosci., Vol. 15, Number 2, June 1990, pp. 83-91. Printed in India. Control of ornithine decarboxylase activity in jute seeds by antizyme MALABIKA PANDIT and BHARATI GHOSH Department of Botany, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Calcutta 700 009, India MS received 8 January 1990; revised 10 May 1990 Abstract. The control of ornithine decarboxylase activity by antizyme was studied during early germination of jute seeds (Corchorus olitorius). When 2 mm of putrescine and spermidine were applied to the germinating medium, the enzyme activity was markedly inhibited (1. 7-fold) during 16 h imbibition. This inhibition could be attributed to the formation of an inhibitory protein termed antizyme. The antizyme was partially purified from jute and barley seedlings. The activity of jute ornithine decarboxylase antizyme was weaker than that of barley. Keywords. Antizyme; ornithine decarboxylase; polyamines; putrescine; spermidine. Introduction In animal (Canellakis et al., 1978), bacteria (Heller et al., 1983a) and certain plants putrescine is formed from ornithine by ornithine decarboxylase (ODC), L-ornithine decarboxylase (EC 4 1 1 17), a rate limiting enzyme for polyamine biosynthesis. Precise regulation of the enzyme has been reported in mammalian systems while in plants the information is scanty. In mammals, ODC is controlled by (i) post translation modification (Russell, 1981), (ii) changes in synthesis and turnover rate (Seely and Pegg, 1983) and (iii) modulation by antizyme (Fujita et al., 1984). In lower plants like Physarium polycephalum there is evidence for phosphorylation of the enzyme (Atmar and Kuehn, 1981). Tyagi et al. (1981) observed post translation modification of ODC in Saccharomyces cerevisiae but in higher plants, there is one report showing the occurrence of ODC-antizyme (Kyriakidis 1983b). ODC activity in most mammalian cells in culture declines rapidly after the addition of putrescine or polyamines by the formation of an inducible protein inhibitor (antizyme) to ODC (Heller et al., 1983b). Antizyme has been isolated from rat liver (Heller et al., 1977), H-35 cells (Fong et al., 1976) and Escherichia coli (Canellakis et al., 1981). The purification and partial characterization of ODC from jute embryos has been already reported from this laboratory (Pandit and Ghosh, 1988). The present paper describes the partial purification of ODC-antizyme and its effect on ODC activity in higher plants. Materials and methods Pyridoxal-5'-phosphate (PLP), dithiothreitol (DTT), Sephadex G-50 were purchased from Sigma Chemical Co., St. Louis, Missouri, USA. DL [l- 14 C]ornithine Abbreviations used: ODC, Ornithine decarboxylase; PLP, pyridoxal-5'-phosphate; DTT, dithiothreitol; Hg 2 Cl 2, mercuric chloride; M r, molecular weight. 83

84 Malabika Pandit and Bharati Ghosh hydrochloride (57 mci/mol) were obtained from Radiochemical Centre, Amersham, UK. All other chemicals used were of analytical grade. Plants Pure line cultivars of jute and barley seeds were obtained from our experimental farm. Germination of seeds Jute and barley seeds after sterilization in 0 1% mercuric chloride (Hg 2 Cl 2 ) were germinated on moistened filter paper in petridishes at 35 ±1 C and 26 ±1 C respectively. Preparation and assay of ODC ODC activity was assayed according to the method of Murakami et al. (1984). Briefly, the embryos were homogenised with 50 mm Tris-HCl buffer (ph 7 6), and the homogenate was centrifuged for 20 min at 26,000 g in a Sorvall refrigerated centrifuge. The supernatant was used as an enzyme source. The assay mixture contained: 50 mm Tris-HCl (ph 7 8), 20 µμ PLP, 5 mm DTT, 0 5 mm DLornithine containing 0 1 µci [l- 14 C]ornithine-HCl and 0 2 ml of enzyme (0 5-2 0 mg of protein) in a volume of 1 ml. After incubating the mixture for 1 h at 37 C, the reaction was terminated by the injection of 0 5 ml of 4 Ν sulfuric acid (H 2 SO 4 ). The released 14 C O 2 was trapped with 0 5 ml of Ν KO H. Protein was estimated by the method of Lowry et al. (1951). One unit of ODC activity is defined as 1 nmol of 14 CO 2 released/h/mg of protein. Preparation and assay of ODC-antizyme The 16 h germinated embryos (10 g fresh wt.) treated with 2 mm putrescine and spermidine were homogenized with homogenizing buffer 50 mm Tris-HCI, ph 7 6, 0 5 mm EDTA and centrifuged at 10,000 g for 20 min. The supernatant was dialyzed overnight against the same buffer at 4 C. The clarified extract was added to purified ODC from 16 h germinated seedlings (Pandit and Ghosh, 1988) and the residual enzyme activity was determined. Appropriate controls were always included. Gel filtration on Sephadex G 50 from jute embryonic axis The supernatant of embryo extract was subjected to 0 50% (NH 4 ) 2 SO 4 and dialyzed for 24 h against the extraction buffer. The dialyzed supernatant after centrifugation was loaded over Sephadex G-50 column (23 1 7) preequilibrated with 50 mm Tris-HCl ph 7 6 containing 0 5 mm EDTA. The resulting fractions (4 ml each) were eluted at a flow rate of 0 3 ml/min.

Preparation and assay of jute ODC and antizyme 85 Molecular weight determination The molecular weight (M r ) of antizyme from jute embryos was estimated by gel filtration in a standardized column of Sephadex G-50 (Whitaker, 1965). ODC- antizyme from barley Antizyme was partially purified according to the method of Kyriakidis (1983a). Protease assay Protease activity was carried out according to Nair et al. (1978). The reaction mixture containing 0 1 Μ potassium phosphate buffer (ph 7 4), casein (100 mg/ml in 0 1 Μ phosphate bluffer ph4), 0.5 ml and enzyme preparation, 0 l-0 2ml was incubated at 37 C for 30 min. The reaction was terminated by 20% trichloroacetic acid and centrifuged at 10,000 g for 15 min. The activity of enzyme was estimated at various time intervals. Heat inactivation The partially purified inhibitory protein, at a concentration of 0 2 mg/ml was split into 5 parts and heated individually on a thermostatic water bath maintained at 10 100 C. Aliquots were withdrawn every 10 min and assayed for inhibitory activity. Results Dose response curve for inhibition of ODC activity The basal ODC activity increased with time during germination of jute seeds and maximum activity was obtained at 16 h. After 16 h germination the activity sharply declined. When the seeds were allowed to germinate in the presence of exogenous putrescine and spermidine, ODC activity was partially inhibited (figure 1) and the inhibition was dependent on the concentration of polyamine. At 1 mm no significant inhibition of ODC activity was observed. However, with increasing polyamine concentration, the inhibition is also increased (figure 2). The germination pattern and vigour of seedlings also changed with various concentration of putrescine and spermidine. However, for further experiments, we used 2 mm putrescine and spermidine for the induction of inhibitory protein where germination percentage and vigour of seedlings are normal. Similarly, inhibition of ODC activity in the presence of polyamines showed that inhibition of ODC is not due to the simple feed back inhibition but for the induction of antizyme (figure 3). Here we used 0 5 mm putrescine and spermidine as maximum inhibition was observed at 0 5 mm. Mixing experiments of purified ODC (1 unit) with increasing amount of dialysed supernatant showed that the maximum amount of inhibition is obtained by 150 µl

86 Malabika Pandit and Bharati Ghosh Figure 1. Effect of 2 mm putrescine and spermidine on ODC activity during germination of jute seeds. The reaction mixture contained 40 mm Tris-HCl buffer ph 7 6, 20 µμ PLP, 5 mm DTT, 1 8nmol of DL [1-14 C] ornithine (Sp. activity 57mCi/mmol) and dialyzed supernatant was incubated for 1 h at 37 C. (O), Control; ( ), treated with spermidine and putrescine. Figure 2. Dose response curve of varying concentrations of polyamine for the inhibition of ODC activity purified from 16 h germinated jute embryos. Jute seeds were treated with different concentrations of polyamine and after 16 h germination antizyme activity was assayed as described in 'materials and methods'. of extract containing 0 2 mg of protein. Such polyamine dependent inhibition was also reported in barley seed (Koromilas and Kyriakidis, 1988). On the other hand no such inhibition of ODC activity was observed by the dialysed supernatant of control extract which suggest that the antizyme was not induced without exogenous addition of putrescine and spermidine. However, antizyme, as a normal component

Preparation and assay of jute ODC and antizyme 87 Figure 3. ODC activity in the presence of putrescine and spermidine. The reaction mixture contained 40 mm Tris-HCl buffer ph 7 6, 20 µμ PLP, 5 mm DTT, l 8 mmol of DL [1-14 C] ornithine (Sp. activity 57mCi/mmol) and dialyzed supernatant was incubated for 1 h at 37 C. (O), Control; ( ) in the presence of 0 5 mm putrescine and spermidine. of H-35 cells, has been reported (Heller et al., 1977). Similar findings have also been reported in wheat leave extract (Smith and Marshall, 1988). Table 1 summarizes the partial purification steps of antizyme. Though several active fractions were eluted in Sephadex G-50 column chromatography, purification of the most active fraction is presented. Further purification was not possible due to its instability. Table 1. Partial purification of antizyme from jute seeds (C. olitorius). Antizyme was partially purified from jute seed using ammonium sulphate saturation and gel filtration on Sephadex G-50. About 19-fold purification of antizyme with a recovery of 39% was achieved. Identification of inhibitory activity To detect the presence of inhibitor in jute seeds, we measured ODC activity with various concentrations of combined putrescine and spermidine. Inhibition may be accounted for by the presence of antizyme in the extract as judged by the following experiments.

88 Malabika Pandit and Bharati Ghosh The inhibitory protein is heat labile at 60 C. About 80% of the activity was lost at the end of 1 h. Trypsin inhibits the antizyme activity by 50 and 8 5 % inhibition at 100 and 200 µg/ml trypsin respectively. The partially purified inhibitory protein was tested for protease activity using both the substrate like casein (hydrolysed) and albumin. No proteolytic activity was detected using phosphate buffer ph 7 4 and ph 4. M r of the antizyme The M r of jute antizyme was judged by the elution profile on Sephadex G-50 and has been estimated to be 25,700 (figure 4). Figure 4. M r - determination by gel filtration. A portion of the partially purified inhibitory protein was chromatographed on a column of Sephadex G-50 (23 1 7 cm) using 0 01 Μ phosphate buffer (ph 7-6) containing 0 2 Μ NaCl with a flow rate of 12 ml/h. The column was standardized by using (1-2 mg) of proteins of known M r. The standard proteins were (1) Rnase-A, (2) cytochrome-c, (3) pepsin and (4) ovalbumin. Titration curve between ODC and antizyme When increasing amounts of antizyme were added to 1 unit of ODC, the activity decreased linearly and the percentage of inhibition by the same amount of jute and barley antizymes are 60 and 90% approximately (figure 5). The titration curve is almost identical to those reported from other systems. Nature of inhibition At different inhibitor concentrations, the inhibitory constant (K I ) was determined which shows that the enzyme was inhibited non competitively with a K I = 1 5 10-3 M(figure 6). Specificity of interaction Inhibition of jute ODC by barley antizyme suggests that there is a non-specific

Preparation and assay of jute ODC and antizyme 89 Figure. 5. Titration curve of ODC purified from 16h germinated jute embryo with antizyme from jute (O) and barley ( ) respectively. Increasing amount of antizyme fractions from column chromatography were added to 1 unit of ODC. The activity was assayed under standard conditions. Figure 6. Effect of antizyme on the Lineweaver-Burk plot of ornithine decarboxylase reaction. Each assay contained 1 unit of ODC supplemented with 27 µμ ( ) 54 µμ ( ) and 81 µμ ( ) antizyme. interaction. Similar observations on acidic antizymes of E. coli inhibiting ODC activity in rat liver (Canellakis et al., 1985) and Tetrahymena pyriformis (Sklaviadis et al., 1985) respectively are available.

90 Malabika Pandit and Bharati Ghosh Discussion The existence of ODC antizyme has been confirmed in germinating jute seeds Similar type of observations were also reported by others when polyamines were added to the culture medium of H-35 cells (Fong et al., 1976), HTC cells (McCann et al., 1977) rat hepatoma cell culture (Kallio et al., 1977) and Hela cells (Branca and Herbst, 1980). The antizyme was partially purified by (NH 4 ) 2 SO 4 saturation and Sephadex G-50 column chromatography. Previous workers have reported M r of 22000 and 26500±2000 respectively for the antizyme from rat liver and neuroblastoma cells (Heller et al., 1977; Canellakis et al., 1985). In the present study the M r of jute antizyme was estimated to be 25,700 from gel filtration data. It has been also observed that antizyme is a potent inhibitor of ODC and inhibits the enzyme in a non-specific way. However, there appears an underlying specificity of interaction of ODC with antizyme in a few systems. It is therefore possible that antizyme forms an inactive complex with the enzyme which would be a means of inactivating ODC and causing rapid degradation (Kyriakidis et al., 1978). The presence of ODC-antizyme in unstimulated H-35 cells and in rat liver coupled with its induction by polyamines provide strong circumstantial evidence that it has some role in modulation of ODC in vivo. In plants the characterization of the nature and function of antizyme in physiological processes remain unexplored. The elucidation of the mechanisms which are responsible for rapid down regulation and turnover of the enzyme durining germination of higher plants is in progress. Acknowledgements Financial help from the Bose Institute was gratefully acknowledged. Authors thank Prof. A. K. Roychoudhury, for kind co-operation. References Atmar, V. J. and Kuehn, G. D. (1981) Proc. Natl. Acad. Sci. USA, 78, 5518. Branca, Α. Α. and Herbst, Ε. J. (1980) Biochem. J., 186, 925. Canellakis, Ε. S., Kyriakidis, D. Α., Heller, J. S. and Pawlak, J. W. (1981) Med. Biol., 59, 279. Canellakis, Ε. S., Heller, J. S., Kyriakidis, D. A. and Chen, K. Y. (1978) Adv. Polyamine Res., 1, 17. Canellakis, Ε., Kyriakidis, D. Α., Rinehart, C. Α., Huang, Α., Panagiotidis, Ε. and Fong, W. F. (1985) Biosci. Rep., 5, 189. Fong, N., Heller, J. S. and Canellakis, Ε. S. (1976) Biochim. Biophys. Acta, 428, 456. Fujita, K., Matsufuji, S., Murakami, Y. and Hayashi, S. (1984) Biochem. J., 218, 557. Heller, J. S., Kyriakidis, Α., Fong, W. F. and Canellakis, Ε. S. (1977) Eur. J. Biochem., 81, 545. Heller, J. S., Rostomily, R., Kyriakidis, A. and Canellakis, Ε. S. (1983a) Proc. Natl. Acad. Sci. USA, 80, 5181. Heller, J. S., Kyriakidis, D. A. and Canellakis, Ε. S. (1983b) Biochim. Biophys. Acta, 760, 154. Kallio, Α., Lofman, Μ., Poso, Η. and Janne, J. (1977) Biochem. J., 177, 63. Koromilas and Kyriakidis, A. (1988) Phytochemistry, 27, 989. Kyriakidis, D. A. (1983a) Adv. Polyamine Res., 4, 427. Kyriakidis, D. A. (1983b) Physiol. Plant., 57, 499. Kyriakidis, Α., Heller, J. S. and Canellakis, E. (1978) Proc. Natl. Acad. Sci. USA, 78, 4699. Lowry, Ο. Η., Rosebrough, Ν. J., Farr, Α. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265. McCann, P. P., Tardif, E. and Mamont, P. S. (1977) Biochem. Biophys. Res. Commun., 75, 948. Murakami, Y., Fujita, K., Kameji, T. and Hayashi, S. (1984) Biochem. J., 217, 573.

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