Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium. Thermus thermophilus.
Molecular and Cellular Biochemistry 195: 55–64, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus A.A. Pantazaki, C.G. Anagnostopoulos, E.E. Lioliou and D.A. Kyriakidis Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece Received 3 October 1997; accepted 5 June 1998
Abstract Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56–292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus. (Mol Cell Biochem 195: 55–64, 1999) Key words: ornithine decarboxylase, antizyme, Thermus thermophilus Abbreviations: ODC – ornithine decarboxylase; Az – antizyme; PLP – pyridoxal-5′-phosphate; Spm – spermine; Spd – spermidine; Put – putrescine
Introduction Polyamines are necessary components of nearly all living cells, although their specific functions have not yet been determined [1, 2]. Polyamine depletion results in reduction of cell growth and even in cell death [3, 4]. The pathway of polyamine biosynthesis begins with the decarboxylation of ornithine to putrescine by ornithine decarboxylase (ODC, L-ornithine carboxylyase, EC 4.1.1.17). The regulation of polyamine biosynthesis is a complex
phenomenon and ODC is a highly regulated enzyme [5–7]. The levels and the activity of ODC can be modulated, positively and/or negatively, at the transcriptional, translational and post-translational level [1, 2, 5, 8]. ODC is regulated post-translationally by covalent modification, degradation, or interaction with an antizyme (Az) inhibitor protein [8]. Antizyme is a non-competitive inhibitor of ODC whose synthesis is induced by polyamines [9, 10]. The inhibition of ODC by Az is reversible and active ODC can be recovered
Address for offprints: D.A. Kyriakidis, Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece
56 from inactive ODC-Az complexes [11, 12]. In addition, there is evidence that Az is involved in ODC degradation of mammalian cells [13–15]. The ODC is degraded by the 26S proteasome in an ATP- and antizyme-dependent manner [16–18]. Furthermore, polyamines have been found to regulate the expression of Az in vitro by inducing ribosomal frameshifting [19]. The Az of E. coli has also been studied extensively [20–21]. In the thermophilic eubacterium T. thermophilus the existence of novel polyamines, such as thermine and thermospermine has been reported [22]. In this organism, with the exception of Clostridium thermohydrosulfuricum [23–24], the concentration of these novel polyamines is higher than normal but the pathway of polyamine biosynthesis has not been investigated. In the present study we report the purification and characterization of ODC as well as its Az of T. thermophilus. Our results demonstrate that in the T. thermophilus cells ODC is metabolically regulated by its Az as shown in some other systems.
structural gene [26] was kindly supplied by Dr. S.M. Boyle (Virginia Tech, USA). Cells were grown in Luria’s broth medium to late log phase. Cells were collected by centrifugation at 6,000 g for 10 min at 4°C, washed in ice cold phosphate buffered saline and kept at –20°C.
Materials and methods
Purification of Thermus thermophilus ODC
Materials
T. thermophilus cells (50 g) were suspended in 125 ml of buffer A (50 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 50 µM PLP, 0.5 mM PMSF and 2 mM β-mercaptoethanol). The cells were lysed by adding lysozyme (1 mg/ml final concentration) and incubated at room temperature for 30 min. DNase I and RNase A were added at a final concentration of 0.1 mg/ml as well as 1% v/v Nonidet P40 and the mixture was stirred for 30 min at 37°C. The mixture was centrifuged at 10,000 × g for 15 min. The 10,000 × g supernatant following lysis of the microorganisms was centrifuged at 100,000 × g for 1 h and the final supernatant was used for the purification of the ornithine decarboxylase. The 100,000 × g supernatant was acidified to pH 6 and after centrifugation at 10,000 × g for 15 min the pH of the supernatant was adjusted to 7.5. Solid ammonium sulfate was added to the supernatant and the pellet was precipitated at 25–60% saturation, dissolved in 70 ml buffer A and dialysed overnight at 4°C against 4 l of buffer A. The dialysed preparation was applied to a DEAE-Biogel A column (1.4 × 20 cm) equilibrated with buffer A. The column was eluted with 10 vol. of buffer A followed by a total 400 ml linear gradient of 0–0.5 M NaCl in buffer A. The active fractions which were eluted at approximately 0.25 M NaCl were pooled and concentrated by ultrafiltration (PM 30, Amicon) to about 10 ml. The enzyme solution from the previous step was applied to a Heparin-Sepharose column (vol. 10 ml) previously equilibrated with buffer A. The active fractions eluted in the flowthrough were pooled and concentrated to 1 ml by ultrafiltration (PM 30, Amicon). The enzyme solution was
Tryptone and yeast extract were purchased from Oxoid. Ammonium sulfate, acrylamide and bis-acrylamide were from Serva. TEMED and ammonium persulfate were from BioRad. Sephacryl S-300 and Protein A-Sepharose CL-4B were from Pharmacia. [8-3H]-5′-GTP, [γ-32P]-ATP and 32P-orthophosphate were purchased from Amersharn. L-[1-14C]-ornithine (sp. activity 50 mCi/mmol) was purchased from Moravek Biochem. Agarose was purchased from BIO-RAD Laboratories (California, USA). ODC antibody was a generous gift from Dr. C. Panagiotidis. All other chemicals were purchased from Sigma.
Bacterial strains, plasmids and growth conditions The T. thermophilus strain HB8 was used in all experiments. Microorganisms were grown at 75°C in a medium containing 0.3% (w/v) yeast extract (Difco), 0.5% (w/v) tryptone (Difco), 0.2% (w/v) NaCl, 0.1% (w/v) D-glucose, 2 µM FeC13, 0.2 mM CaCl2 and 1 mM MgCl2. The pH was adjusted to 7.0 with KOH. Growth was monitored by measuring the turbidity at 600 nm. The bacteria were harvested at the end of the logarithmic phase by centrifugation at 6,000 g for 10 min. Cells were washed twice with 0.9% (w/v) NaCl. The final yield was about 5 g of wet cells per liter of culture medium. The E. coli K-12 strain MG-1655 (F-, λ-) [25] was also used. Plasmid pODC, a pBR322 plasmid containing the ODC
Purification of E. coli ODC ODC was purified to apparent homogeneity as described previously [27] from an ODC overproducing E. coli K12 strain MG1655 (F-, λ-) transformed with plasmid pODC (a pBR322 plasmid carrying the ODC gene). The ODC purification protocol [27], including streptomycin sulfate precipitation of the nucleic acids, 39–51% ammonium sulfate fractionation, heat treatment at 62°C for 10 min and chromatography on a DEAE-Biogel A and a Sephacryl S-300 column was modified by adding as the final stage of purification a Biogel P-100 column.
57 applied to a Sephadex G-150 column (240 ml volume) equilibrated with buffer A. The active fractions of this column were pooled and dialysed overnight against buffer B (0.3 M sodium phosphate pH 6.8, 0.1 mM EDTA, 50 µM PLP, 0.5 mM PMSF and 2 mM β-mercaptoethanol) and applied onto a Phenyl-Sepharose column (30 ml volume), equilibrated in the same buffer. The column was washed with a 120 ml linear gradient of 0.3 M– 5 mM of phosphate buffer pH 6.8 discharging the bulk of the protein, and consecutively with a 300 ml linear gradient 0–70% ethyleneglycol in buffer C (5 mM sodium phosphate pH 6.8, 0.1 mM EDTA, 50 µM PLP, 0.5 mM PMSF and 2 mM β-mercaptoethanol). The active enzyme was eluted at approx. 60% ethyleneglycol. The active fractions of this column were combined and applied to a Lysine-Sepharose column (0.5 × 8 cm). The column was washed with 3 vol of buffer A and sequentially eluted by a total 80 ml linear gradient of 0–0.6 M NaCl in buffer A. The active fractions which were eluted at approximately 0.35 M NaCl, were pooled and concentrated by ultrafiltration (PM 30, Amicon) to approximately 1 ml. The enzyme solution was dialysed overnight against buffer D (50 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.5 mM PMSF, 2 mM β-mercaptoethanol and 0.1% Tween 80) and applied to a Pyridoxamine-Affi-Gel 10 column (vol 10 ml) prepared according to the manufacturer’s instructions (Pharmacia) and previously equilibrated with buffer D. The column was eluted with 3 vol buffer D followed by 3 vol stepwise elution with 100 mM PLP in buffer D. The antizyme of E. coli was purified as previously described [20].
Immunoprecipitation of T. thermophilus antizyme and polyacrylamide gel electrophoresis T. thermophilus cells were suspended in 5 volumes of ODC assay buffer, disrupted by sonication, centrifuged and the antibody was added to the supernatant. After 1 h of incubation in ice, Protein A-Sepharose beads were added. After a further 1 h incubation in ice, the beads were washed 3 times with the same buffer. The beads were then suspended in SDS-PAGE loading buffer, incubated in boiling water for 3 min, so as to elute the proteins. SDS-polyacrylamide slab gel electrophoresis was performed as described by Laemmli [28] for 30 min at 30 volts and for 3–4 h at 80 volts.
Blotting of proteins onto membranes and staining with antibody The blotting of the proteins onto nitrocellulose membranes and the staining with antibody were performed as described by Harlow and Lane [29].
Purification of the (GST) glutathione S-transferaseantizyme fusion protein. The GST-AZ fusion protein was purified as described previously [30]. Pyridoxamine-Affi-gel 10 preparation
Pyridoxamine-Affi-gel 10 preparation Pyridoxamine-Affi-gel 10 was prepared according to the manufacturer’s instructions [Affinity Chromatography (1983) Pharmacia Fine Chemicals AB, Sweden].
Pyridoxamine-Affi-gel 10 was prepared according to the manufacturer’s instructions [Affinity Chromatography (1983) Pharmacia Fine Chemicals AB, Sweden].
Results Assay for ornithine decarboxylase and antizyme Enzyme assays were performed as previously described [12, 20]. The optimal conditions of assaying ODC of T. thermophilus were found to be 50 mM Tris-HCl buffer pH 7.5 containing 2.5 mM DTT and 50 µM PLP at 65°C. The Az activity was assessed by measuring the inhibition of a known amount of ODC. Binding of the E. coli ODC to Sepharose beads The binding of ODC to CNBr-activated Sepharose CL-4B beads was performed according to the manufacture (Pharmacia Chromatographic Manual).
Localization and extraction of ornithine decarboxylase In T. thermophilus the ODC activity was located as 53% in the cytosolic fraction (100,000 × g supernatant for 1 h) with the remaining activity in the 100,000 × g pellet. The total recovery was 97% in triplicate experiments. Since half of the activity remained in the pellet, various reagents were tested to extract ODC from the 100,000 × g pellet in a soluble form; increased concentration of salts (0.5–2.0 M NaCl), chaotropic agents (0.1 M KSCN), non ionic detergents (0.5–2.0% v/v Nonidet P40, 1% v/v Triton X-100), chelating agents (2.0–5.0 mM EDTA), DNase I, lipase and phospholipase C were tested to extract ODC from the pellet. Among them, Nonidet P40 (1% v/v) in buffer A
58 was the most effective in solubilizing the enzyme. The remaining activity in the 100,000 × g pellet was released by a second extraction with this buffer.
Purification of Thermus thermophilus ODC The purification scheme developed for ODC is summarized in Table 1. A calculation of the yield and purification of ODC showed 4.3% recovery with 2330-fold purification. This preparation showed one protein band in SDS-polyacrylamide gel electrophoresis stained either by silver nitrate or with antibodies raised against purified E. coli ODC (Fig. 1). The specific activity of our preparation was two orders of magnitude lower than those of E. coli [27] but very close to those reported for Physarum polycephalum [31], Saccharomyces cerevisae [32] and Tetrahymena pyriformis [33]. It should be noted that the S. cerevisae ODC has been shown by Fonzi and Sypherd [34] to have much higher specific activity than reported earlier by Tyagi et al. [32].
Physical and catalytic properties of ODC The molecular mass of the enzyme as estimated by its mobility on a Sephadex G-150 column was 135 kDa. In SDS-polyacrylamide gel electrophoresis, the same preparation gave one major band of 68 kDa. These findings lead to the conclusion that the enzyme has a dimeric form. The purified enzyme acts optimally at pH 7.5 which is very close to the optimum pH of the E. coli ODC. The enzyme was pyridoxal-5′-phosphate-dependent. The Km for L-ornithine as determined from a Lineweaver-Burk plot was 0.5 mM (data not shown). When purified ODC was incubated with increasing amounts of calf intestine alkaline phosphatase bound to agarose (30 units/mg) a progressive conversion of active Table 1. Summary of the purification procedure of T. thermophilus ODC Steps
Total Total protein activity
Crude extract 31 6482.0 Acidification 1845.0 5925.0 Ammonium1018.0 4530.0 Sulfate (25–60%) DEAE-Biogel A 228.6 845.0 Heparin-Sepharose 69.7 2100.0 Sephadex-G 150 25.8 935.0 Phenyl-Sepharose 2.2 620.0 Lysine-Sepharose 0.1 414.0 Pyridoxamine-Affi0.06 280.0 Gel-10
Spec. act. (units/mg) 2.0
Purification Yield (fold) (%)
4.4
1.0 1.6 2.1
100.0 91.4 69.8
16.8 30.1 36.2 282.3 4140.0 4666.0
8.4 15.0 18.1 141.1 2070.0 2333.0
59.3 32.4 14.4 9.5 6.4 4.3
One unit of enzyme activity = 1 nmol of CO2 released/1 h.
Fig. 1. SDS-polyacrylamide gel electrophoresis of purified ODC. Purified ODC (1 µg) was subjected to SDS-polyacrylamide gel electrophoresis in 10% polyacrylamide gel. (a) The gel was stained with silver nitrate. The standard protein markers used were: (1) Myosin (H-chain) 200, (2) Phosphorylase b 97.4, Bovine serum albumin 68, (4) Ovalbumin 43, (5) Carbonic anhydrase 29, (5) β-Lactoglobulin 18.4 and (6) Lysozyme 14.3 kDa. (7) Bb, bromophenol blue. (b) Immunostaining of the nitrocellulose membrane with ODC-antibodies raised against purified ODC from E. coli.
ODC to inactive ODC was obtained (Fig. 2). The type of curve was biphasic, indicating that removal of phosphate groups with low amounts of alkaline phosphatase caused abrupt inhibition of ODC activity (–80%), while further inhibition required high amounts of alkaline phosphatase. When native purified or dephosphorylated ODC were subsequently incubated with increasing concentrations of nucleotides, a progressive parallel increase in the two forms of ODC activity was obtained (Fig. 3). Uracil, adenine and cytidine nucleotides were also tested for activation of ODC activity, and similar results to guanine nucleotides were obtained with all tested nucleotides and the order of ODC activation was NTP>NDP>NMP (data not shown). The differential in vivo effect of polyamines in different cell lines and in bacteria through the appearance of antizyme was well documented [1, 2, 10, 11, 13]. In addition mammalian ODC is not inhibited in vitro by polyamines [1, 8]. Putrescine, added to the in vitro assays of Tetrahymena pyriformis ODC, causes measurable inhibition at concentrations above 10–4 M [33]. Contrary to that the bacterial ODC is very sensitive to polyamines added in the in vitro assays. Therefore, it was interesting to look for the sensitivity of the highly purified ODC of T. thermophilus to polyamines. Addition of putrescine in vitro to the purified ODC caused slight inhibition, whereas spermine and spermidine activated ODC about 140 and 40%, respectively (Fig. 4).
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Fig. 2. Effect of increasing concentrations of alkaline phosphatase bound to agarose on the activity of T. thermophilus ODC. Pure ODC (5 units) was incubated for 30 min with increasing concentrations of alkaline phosphatase bound to agarose. The mixtures were then centrifuged and the reaction started with the addition of 0.023 µmol of 14C-L-ornithine in the supernatants.
A thermo-stability curve was established for the highly purified enzyme by preincubation for 15 min at each of the temperatures, leading to 85% loss of ODC activity at 75°C and more than 95% loss when the temperature was increased over 85°C. It is known that partially purified ODC from different sources exhibited a broad substrate specificity, decarboxylating several amino acids [33, 35]. Substrate competition studies with lysine, arginine and histidine showed that the two former amino acids inhibited the enzyme in a competitive manner, while histidine did not have any effect (data not shown). It was reported that phospho-amino acids inhibit non competitively the T. pyriformis, rat liver and E. coli ODC [33]. Phospho-serine activated the highly purified enzyme linearly and at a concentration of 2 mM caused a 1.5-fold increase in activity, phospho-threonine or phospho-tyrosine activated ODC to a lower degree (Fig. 6). The highest activation was obtained with 0.5 mM or 1.0 mM of phospho-threonine or phospho-tyrosine, respectively.
growth of T. thermophilus was not significantly affected, as determined by turbidity at 600 nm.
Effect of vitamin B6 on the induction of ODC activity in vivo
Purification of Thermus thermophilus antizyme
Supplementing the normal growth medium of T. thermophilus with vitamin B6 (pyridoxine) at concentrations of 10 or 80 mg/l, resulted in an increase of ODC activity by 178 and 283%, respectively (Table 2). When ornithine (10 mg/l) and vitamin B6 (80 mg/l) were added, ornithine decarboxylase activity was stimulated to 204%. Under these conditions the
Effect of polyamines on the growth and specific activity of the ODC of Thermus thermophilus Putrescine at 5 mM concentration delayed the growth of T. thermophilus. This effect was more prominent when spermidine (2.5 or 5.0 mM) was added to the growth medium. Addition of spermine, even at 0.5 mM, completely inhibited growth. A peak of ODC activity appears at 6 h of growth (log phase), which drops when the cells reach the stationary phase. Low concentrations of polyamines resulted in an increase of this peak of ODC activity. This effect was evident in the order Spm>Spd>Put. In contrast, high concentrations of polyamines added to the growth medium resulted in a decrease of the specific activity of ODC, due to the appearance of Az as previously described [9].
T. thermophilus cells were grown in the presence of 2.5 mM putrescine and 2.5 mM spermidine for 20 h at 75°C. The cells were collected by centrifugation, suspended in 5 vol of Tris-HCl pH 8.0, 50 µM pyridoxal-5′-phosphate, disrupted by sonication and centrifuged. The supernatant was applied onto an ODC-Sepharose column (prepared as described in Materials and methods) and the column was washed with the
60 thermophilus Az by immunoprecipitation using antibody against the E. coli Az. The antibody was added in the total cellular extract, incubated on ice for 1 h, protein A-Sepharose beads were added and the mixture was incubated further for 1 h. The beads were washed three times with the same buffer and the Az was eluted using SDS-PAGE sample buffer and boiling (Fig. 8B). Due to the harsh conditions used for the elution the Az was not active, although the yield was much improved.
Cross-interaction of ODC and Az from Thermus thermophilus and E. coli The antizyme of T. thermophilus can inhibit not only the homologous ODC of T. thermophilus but the ODC of E. coli as well, although to a lower degree (Fig. 9a). The inverse experiment which was also performed showed that the Az of E. coli also inhibited T. thermophilus ODC as well (Fig. 9b).
A GST-antizyme fusion protein does not inhibit ODC A fusion protein consisting of the glutathione S-transferase and aminoacids 56-292 of the E. coli antizyme was checked
Fig. 3. Activation of ODC by nucleotides. (A) Reverse activation of phosphatase-treated ODC: Pure ODC (5 units) was incubated with 5 units of alkaline phosphatase bound to agarose for 30 min. After removal of the alkaline phosphatase by centrifugation, increasing concentrations of nucleotides were added and the reaction was continued for 60 min with addition of 0.023 µmol of 14C-L-ornithine; (B) Activation of native ODC: Pure ODC (2 units) was incubated with increasing concentrations of nucleotides and the reaction was carried out for 60 min with addition of 0.023 µmol of 14C-L-ornithine. GTP (■); UTP (●); ATP (▲); GMP (▼); Adenosine (◆).
same buffer. The Az was eluted as a single peak by 10% saturated ammonium sulfate (Fig. 7). The Az fractions were pooled and subjected to SDS-PAGE, which showed one protein band at about 50 kDa (Fig. 8Aa). The same band appeared after the protein bands were transferred onto nitrocellulose membrane and stained with antibody against E. coli Az (Fig. 8Ab). It should be noted that E coli Az has a molecular mass of 49500 [20]. Because of the low yield of this procedure (less than 1 µg of Az/g of wet cells), we attempted the purification of T.
Fig. 4. In vitro effect of polyamines to the highly purified T. thermophilus ODC. Increasing amounts of polyamines, from a stock solution adjusted to the optimum pH in assay buffer, were added to purified T. thermophilus ODC.
61 neither of E. coli nor of T. thermophilus indicating that the inhibitory site of E. coli Az is either not located in this fragment or participates in the tertiary structure necessary for binding of Az to ODC.
Discussion
Fig. 5. Thermal stability curve for ODC. Purified ODC was incubated for 15 min at each temperature point and a sample was assayed by adding 14 C-L-ornithine as described in Materials and methods.
for inhibition on ODC activity. For the production of this protein, a 712-bp PvuII-EcoRI fragment (bp 294-1002 of the antizyme gene) was ligated to plasmid pGEX-3X, which had been cut with EcoRI and SmaI. The resulting recombinant plasmid produced a GST-Az fusion protein which contained amino acids 56–292 of the E. coli Az, did not inhibit the ODC
The purified ODC of T. thermophilus has an apparent native Mr of approximately 135000, with a subunit of 68000 as determined by SDS electrophoresis and immunostaining. A subunit of Mr 55000 has been reported for the enzyme of T. vaginalis [35] and of germinated barley seeds [36]. A subunit of Mr 53000 has been reported for the mouse and rat liver tissue ODC [37]. It should be noted that ODC purified from various eucaryotes such as Saccharomyces cerevisae [32], Tetrahymena pyriformis [33] and Physarum polycephalum [31] shows small differences to the mammalian ODC in molecular mass. High concentration of polyamines is known to have a cytostatic effect on bacteria [3, 4]. Increasing the concentration of polyamines in the growth medium of T. thermophilus also resulted in a delay or even in complete inhibition of cell growth. It should be noted that these physiological polyamines are synthesized only in minute amounts by this microorganism [22]. Low concentrations of polyamines result in an in vivo increase of ODC specific activity, in the order putrescine < spermidine < spermine, whereas high concentrations result in a decrease of the specific activity. It is possible that low
Fig. 6. Effect of phospho-aminoacids on ODC activity. Increasing amounts of P-Ser, P-Thr and P-Tyr from a 20 mM stock solution in assay buffer, adjusted to optimum pH were added to the purified T. thermophilus ODC.
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Fig. 7. Purification of the T. thermophilus Az by ODC-Sepharose affinity column. The supernatant of lysed cells was loaded onto the column (equilibrated in Tris-HCl pH 8.0 + pyridoxal-5′ phosphate), the column was washed and the Az was eluted by 10% saturated (NH4)2S04.
concentrations of polyamines induce the production of an activator of the ODC, whereas high concentrations induce Az production. Interesting is the observation that purified ODC can be activated in vitro by spermine and spermidine. Therefore, the effect of spermidine and spermine could be compared with the effect of thermo-spermidine and thermospermine, the polyamines which are found at high concentrations in T. thermophilus [22]. The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column, and by immunoprecipitation using antibody against the E. coli antizyme, and showed similar physicochemical properties as that of the E. coli Az [12, 13, 20]. The gene of the E. coli antizyme has been isolated in a 6.4 kb fragment from a genomic library [38]. This fragment contains two open reading frames (ORF), the one of which codes for the Table 2. Effect of Vitamin B6 on the induction of ODC in vivo Composition of medium
% ODC activity
Normal +B6 (10 mg/l) +B6 (80 mg/l) +B6 (80 mg/l)+ornithine (10 mg/l)
100 178 283 204
T. thermophilus was grown in the normal medium (50 ml) supplemented at zero time with 10 or 80 mg/l of vitamin B6 (pyridoxine). Cells were collected at the end of the logarithmic phase, lysed and ODC activity was assayed as described. Growth was monitored by measuring the absorbance at 600 nm in a Perkin-Elmer spectrophotometer.
Fig. 8. SDS-PAGE of purified Az. (A) The fractions corresponding to the peak eluted from the ODC-Sepharose column were pooled, concentrated and subjected to SDS-PAGE as well as immunoblotting; (a) staining of purified Az by silver nitrate and (b) staining of the Az by antibody against the E. coli Az; (B) The Az was immunoprecipitated by the addition of Az antibody to the cellular extract. The complex was bound to Protein A-Sepharose beads, and the Az was eluted using SDS-PAGE sample buffer, transferred onto nitrocellulose membranes and stained with E. coli Az antibody. Numbers on the right indicate molecular mass in kDa of the standard protein markers used as in Fig. 1.
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Fig. 9. Titration of E. coli and T. thermophilus ODC (a) by T. thermophilus Az or (b) by E. coli Az, Increasing amounts of either E. coli or T. thermophilus Az were added to a constant amount of either T. thermophilus or E. coli ODC and the percentage of inhibition of the ODC activity in each case was measured.
antizyme and the second was found to be homologous to the modulator protein of the two component regulator systems of bacteria. When the appropriate environmental signal arrives at the cell this protein is phosphorylated and the phosphate is subsequently transferred to another protein, the effector, which is responsible for the cellular response to the environmental signal. The sequence of the antizyme was found to show homology with the effector proteins. Because of this, we tested the effect of incubating the antizyme of T. thermophilus and E. coli with phosphatases, on their ability to inhibit ODC. Neither calf intestine alkaline phosphatase, nor human prostate or wheat acid phosphatases affected the antizyme activity. This may indicate that either the Az is not phosphorylated or that dephos-
phorylation does not affect its activity. It has also been shown that the effector proteins of the two component regulator systems are phosphorylated on an aspartate residue [39, 40], but the phosphatases tested in this study do not cleave this type of bond. The antizyme of E. coli can inhibit the ODC of T. thermophilus and vice versa. Finally the fusion protein of glutathione S-transferase with amino acids 56–292 of the E. coli antizyme could not inhibit the ODC activity of E. coli or T. thermophilus, indicating that the binding site of Az with ODC may not be located in this fragment of the Az. The exact role of Az as well as its ability to react and inactivate ODC in the bacterial system remains to be determined. The above question needs to be addressed at the biochemical as well as at the genetic level.
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References 1. Canellakis ES, Viceps-Madore D, Kyriakidis DA, Heller JS: The regulation and function of ornithine decarboxylase and of the polyamines Curr Top Cell Reg 15: 155–202, 1979 2. Pegg AE: Recent advances in the biochemistry of polyamines in eukaryotes. Biochem J 234: 249–262, 1986 3. Tabor CW, Tabor H: Polyamines. Ann Rev Biochem 53: 749–790, 1984 4. Tabor CW, Tabor H: Polyamines in microorganisms. Microbiol Rev 49: 81–89, 1985 5. Davis RH, Morris DR, Coffino P: Sequestered end products and enzyme regulation: The case of ornithine decarboxylase. Microbiol Rev 56: 280–290, 1992 6. Kameji T, Hayashi S, Koshimo K, Kakinuma Y, Igarashi K: Multiple regulation of ornithine decarboxylase in enzyme-overproducing cells Biochem J 289: 581–586, 1993 7. Hayashi S, Murakami Y, Matsufuji S: Ornithine decarboxylase antizyme: A novel type of regulatory protein TIBS 21: 27–30, 1996 8. Hayashi S, Canellakis ES: Ornithine decarboxylase antizymes. In: S. Hayashi (ed). Ornithine Decarboxylase: Biology, Enzymology and Molecular Genetics. Pergammon Press, New York, 1989, pp 47–58 9. Fong WF, Heller JS, Canellakis ES: The appearance of an ornithine decarboxylase inhibitor protein upon the addition of putrescine to the cell cultures. Biochem Biophys Acta 428: 456–465, 1976 10. Heller JS, Fong WF, Canellakis ES: Induction of a protein inhibitor to ornithine decarboxylase by the end products of its reaction. Proc Natl Acad Sci USA 73: 1858–1862, 1976 11. McCann PP, Tardif C, Mamont PS: Regulation of ornithine decarboxylase by ornithine decarboxylase-antizyme in HTC cells. Biochem Biophys Res Commun 75: 948–954, 1977 12. Kyriakidis DA, Heller JS, Canellakis ES: Modulation of ornithine decarboxylase activity in Escherichia coli by positive and negative effectors. Proc Natl Acad Sci USA 75: 4699–4703, 1978 13. Canellakis ES, Hayashi S: The role of antizyme in the regulation of ornithine decarboxylase activity in eukaryotic cells. In: U Bachrach, YM Heimer (eds). The Physiology of Polyamines. CRC, Boca Raton, FL, Vol. 1, 1989, pp 315–330 14. Murakami Y, Matsufuji S, Miyazaki Y, Hayashi S: Destabilization of ornithine decarboxylase by transfected antizyme gene expression in hepatoma tissue culture cells. J Biol Chem 267: 13138–13141, 1992 15. Li X, Coffino P: Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase. Mol Biol 14: 87–92, 1994 16. Murakami Y, Matsufuji S, Kameji T, Hayashi S, Igarashi K, Tamura T, Tanaka K, Ichihara A: Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature 360: 597–599, 1992 17. Murakami Y, Matsufuji S, Tanaka K, Ichihara A, Hayashi S: Involvement of the proteasome and antizyme in ornithine decarboxylase degradation by reticulocyte lysate. Biochem J 295: 305–308, 1993 18. Bercovich Z, Kahana C: Involvement of the 20S proteasome in the degradation of ornithine decarboxylase. Eur J Biochem 213: 205–210, 1993 19. Rom E, Kahana C: Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame shifting. Proc Acad Natl Sci USA 91: 3959–3963, 1994 20. Heller JS, Kyriakidis DA, Canellakis ES: Purification and properties of the antizymes of Escherichia coli to ornithine decarboxylase. Biochim Biophys Acta 760: 154–162, 1983
21. Panagiotidis CA, Canellakis ES: Comparison of the basic Escherichia coli antizyme 1 and antizyme 2 with the ribosomal proteins S20/L34. J Biol Chem 259: 15025–15027, 1984 22. Oshima TE: Novel polyamines in Thermus thermophilus: Isolation, identification, and chemical synthesis. Meth Enzymol 94: 401–411, 1983 23. Poso H, Paulin L: Ornithine decarboxylase activity from an extremely thermophilic bacterium, Clostridium thermohydrosulfuricum. Adv Polyamine Res 4: 489–494, 1983 24. Paulin L, Poso H: Ornithine decarboxylase activity from an extremely thermophilic bacterium, Clostridium thermohydrosulfuricum. Effect of GTP analogues on enzyme activity. Biochim Biophys Acta 742: 197–205, 1983 25. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S: Construction and characterization of new cloning vehicles. II. A multipurpose cloning system Gene 2: 95–113, 1997 26. Boyle SM, Markham GD, Hafner EW, Wright JM: Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli. Gene 30: 121– 136, 1984 27. Huang SC, Panaglotidis CA, Canellakis ES: Transcriptional effects of polyamines on ribosomal proteins and on polyamine-synthesizing enzymes in Escherichia coli. Proc Natl Acad Sci USA 87: 3464–3468, 1990 28. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970 29. Harlow E, Lane D: Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, 1988 30. Smith DB, Johnson KS: Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 31–40, 1988 31. Barnett GR, Kazarinoff MN: Purification and properties of ornithine decarboxylase from Physarum polycephalum. J Biol Chem 259: 179– 183, 1984 32. Tyagi AK, Tabor CW, Tabor H: Ornithine decarboxylase from Saccharomyces cerevisiae. J Biol Chem 256: 12156–12163, 1981 33. Sklaviadis TK, Georgatsos JG, Kyriakidis DA: Purification and properties of ornithine decarboxylase from Tetrahymena pyriformis. Biochem Biophys Acta 831: 288–296, 1985 34. Fonzi WA, Sypherd PS: The gene and the primary structure of ornithine decarboxylase from Saccharomyces cerevisiae. J Biol Chem 262: 10127–10132, 1987 35. Yarlett N, Goldberg B, Moharrami MA, Bacchi CJ: Trichomonas vaginalis: Characterization of ornithine decarboxylase. Biochem J 293: 487–493, 1993 36. Koromilas AE, Kyriakidis DA: Purification and properties of ornithine decarboxylase from germinated barley seeds. Phytochemistry 27: 989– 992, 1988 37. Seely JE, Poso H, Pegg AE: Purification of ornithine decarboxylase from kidneys of androgen-treated mice. Biochemistry 21: 3394–3399, 1982 38. Canellakis ES, Paterakis AA, Huang S-C, Panagiotidis CA, Kyriakidis DA: Identification, cloning and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli. Proc Natl Acad Sci USA 90: 7129–7133, 1993 39. Bourret RB, Borkovich KA, Simon MI: Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu Rev Biochem 60: 401–441, 1991 40. Saier MH, Wu LF, Reizer J: Regulation of bacterial physiological processes by three types of protein phosphorylating system. Trends Biochem Sci 15: 391–395, 1990
