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Agmatine deiminase from maize shoots: purification and properties
Phytochemistry 56 (2001) 643±647
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Agmatine deiminase from maize shoots: puri®cation and properties Hiroshi Yanagisawa * Biology Department, Aichi University of Education, Igaya, Kariya, Aichi 448-8542, Japan Received 18 July 2000; received in revised form 8 November 2000
Abstract Agmatine deiminase was puri®ed to a speci®c activity of 537 nkat/mg protein using an improved procedure. The recovery was 47% and the enzyme was homogeneous and remarkably stable. The molecular mass of the enzyme as determined by gel ®ltration was 75 kDa, and SDS-PAGE suggests that the enzyme is a hetero climer composed of subunits of 43.5 and 44 kDa. The Km for agmatine was 12 mM and arcaine was shown to be a potent competitive inhibitor of the enzyme, with a Ki of 3.3 mM. The enzyme does not have either putrescine synthase activity or the activities of its components ornithine and putrescine transcarbamylase. These results distinctly demonstrate that agmatine deiminase is dierent from putrescine synthase. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Zea mays; Maize; Agmatine deiminase; Putrescine synthase; Enzyme puri®cation
1. Introduction Polyamines are synthesized from arginine and ornithine in higher plants (Smith, 1990). In the arginine pathway, agmatine deiminase (EC 3.5.3.12) hydrolyzes agmatine to N-carbamoylputrescine and ammonia (Smith, 1969). Agmatine deiminase has been puri®ed to homogeneity from maize shoots (Yanagisawa and Suzuki, 1981), rice (Chaudhuri and Gosh, 1985), and soybean (Park and Cho, 1990). On the other hand, putrescine synthase has been puri®ed to homogeneity from grass pea (Srivenugopal and Adiga, 1981) and cucumber (Prasad and Adiga, 1986). Putrescine synthase has activities similar to those of agmatine deiminase, putrescine transcarbamylase, ornithine transcarb amylase, and carbamate kinase in a single polypeptide, and converts agmatine and ornithine to ammonia, putrescine, and citrulline in the presence of inorganic phosphate. Prior to this study, little was known about the kinetics of agmatine deiminase, although it was reported in studies with soybean that this enzyme was a monomer similar to putrescine synthase. However, it was
* Tel.: +81-0566-26-2646; fax: +81-0566-26-2310. E-mail address: [email protected] edu.ac.jp
uncertain whether agmatine deiminase was putrescine synthase itself or clearly dierent from putrescine synthase. In this paper, the characteristics of agmatine deiminase puri®ed from maize shoots by an improved procedure were examined, and compared with those of agmatine deiminase and putrescine synthase from other plants. 2. Results and discussion 2.1. Agmatine deiminase assays Agmatine deiminase activity was previously determined by measuring the ammonia released from agmatine in Conway microdiusion units with Nessler reagent (Yanagisawa and Suzuki, 1981); however, this procedure is insensitive. Moreover, ammonia is released from agmatine by Cu-containing amine oxidase in higher plants, and the estimation of agmatine deiminase activity by measuring the ammonia released from agmatine with extracts including Cu-containing amine oxidase is not accurate. Cu-containing amine oxidase has been detected in several species of leguminous and dicotyledonous plants (Suzuki et al., 1990), and recently in monocotyledonous plants including maize (Cogoni et al., 1990; Suzuki and Hagiwara, 1993). Therefore,
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00491-X
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H. Yanagisawa / Phytochemistry 56 (2001) 643±647
agmatine deiminase activity was determined by measuring N-carbamoylputrescine by the diacetyl monoxime method (Boyde and Rahmatullah, 1980). When measuring the enzyme activity of plant extracts and in an early puri®cation step that involved both agmatine deiminase and Cu-containing amine oxidase, 1 mM sodium diethyldithiocarbamate was added to the reaction mixture. This reagent is a potent inhibitor of Cu-containing amine oxidase and did not in¯uence either the enzyme activity or the colorimetric reaction. 2.2. Eect of dark and light on activities of agmatine deiminase from maize shoots Agmatine deiminase activities of maize shoots grown for 7 days in the dark and light at 25 C were 0.25 and 0.28 nkat/shoot, respectively. No signi®cant dierence was found between the enzyme activities of maize shoots grown in the dark and light for 7 days.
2.4. Kinetics of agmatine deiminase The enzyme reactions were linear with respect to time and enzyme concentrations under standard assay conditions. The optimal pH of the enzyme puri®ed by the improved procedure was 6.5, with 85% of the maximum activity at pH 6.0 and 8.0. The enzyme showed a Michaelis±Menten-type saturation curve. A Km for agmatine of 12 mM was obtained from double-reciprocal plots (Fig. 1). It has been reported that the Km values for agmatine of the enzymes previously puri®ed from maize shoots (Yanagisawa and Suzuki, 1981) and soybean axes (Park and Cho, 1990) were 0.19 and 5 mM, respectively. The high Km values reported previously might be due to an inaccurate estimation of the enzyme activity and the long reaction time. The dierence in the speci®city of agmatine against agmatine
2.3. Puri®cation of agmatine deiminase Agmatine deiminase from maize shoots grown for 7 days in the dark was puri®ed by a DE52 batchwise method, DE52 column chromatography, Sephadex G100 column chromatography, and agmatine-epoxySepharose 6B column chromatography. The puri®cation procedure was largely the same as that described previously (Yanagisawa and Suzuki, 1981), but with the following major modi®cations: 1 mM phenylmethyl sulfonyl ¯uoride (PMSF) and EDTA were added to the extraction buer; and the anity adsorbent was changed from agmatine±succinic acid-hexamethylenediamineSepharose 4B to agmatine-epoxy-Sepharose 6B. The enzyme was puri®ed to a speci®c activity of 537 nkat/mg protein, and obtained in 47% yield (Table 1). The enzyme was previously puri®ed to a speci®c activity of 230 nkat/mg protein and lost 30% of its original activity after 3 days at 4 C (Yanagisawa and Suzuki, 1981). However, the enzyme puri®ed with the improved procedure was stable for two months at 4 C without any loss of activity. The increase in speci®c activity and the outstanding stability of the enzyme suggests that the enzyme puri®ed with the improved procedure has not been modi®ed during puri®cation.
Fig. 1. Competitive inhibition of agmatine deiminase from maize shoots by arcaine. Control (closed circles), in the presence of 50 mM arcaine sulfate (open circles): 50 ng of the enzyme was used.
Table 1 Puri®cation of agmatine deiminase from maize shoots Puri®cation step
Total protein (mg)
Total activity (nkat)
Speci®c activity (nkat/mg)
Puri®cation (fold)
Yield (%)
1. Crude extract 2. Eluate from DE52 3. Eluate from DE52 column 4. Eluate from Sephadex G-100 column 5. Eluate from anity column
603 90.5 18.8 2.61 0.0944
107 92.9 87.1 68.2 50.7
0.178 1.03 4.63 26.2 537
1 5.75 26 147 3010
100 86.4 81 63.5 47.2
H. Yanagisawa / Phytochemistry 56 (2001) 643±647
deiminase and putrescine synthase could not be determined, since the Km for agmatine of putrescine synthase has not yet been reported. Arcaine, an analogue of agmatine, is reportedly a potent inhibitor of the enzyme (Smith, 1969; Yanagisawa and Suzuki, 1981), and this inhibition is competitive, with a Ki for arcaine of 3.3 mM (Fig. 1). It is unclear whether agmatine deiminase puri®ed from soybean and rice and putrescine synthase would also be inhibited by arcaine. 2.5. Subunit structure of agmatine deiminase The molecular mass of enzyme puri®ed by the improved procedure was determined to be 75 kDa by the gel ®ltration method with Sephadex G-100 column chromatography. SDS-PAGE of the enzyme showed very close polypeptides of 43.5 and 44 kDa (Fig. 2). Sugar moieties were not found in the enzyme as evidenced by the phenol sulfuric acid method. These results suggest that the enzyme is a hetero dimer, although those from maize (Yanagisawa and Suzuki, 1981) and rice (Chaudhuri and Gosh, 1985) were reported to be dimers, and that from soybean was reported to be a monomer (Park and Cho, 1990).
645
2.6. Putrescine synthase activity Putrescine synthase, puri®ed to homogeneity from grass pea (Srivenugopal and Adiga, 1981) and cucumber (Prasad and Adiga, 1986), exhibited agmatine deiminase activity of 0.39 and 0.73 nkat/mg protein, respectively. This enzyme was highly unstable: grass pea putrescine synthase lost its component activities within 48 h at 4 C after puri®cation. Agmatine deiminase puri®ed from maize did not have the activities of putrescine synthase (citrulline formation), putrescine transcarbamylase (Ncarbamoylputrescine formation), or ornithine transcarbamylase (citrulline formation). These results distinctly demonstrate that agmatine deiminase diers from putrescine synthase. 3. Experimental 3.1. Chemicals N-Carbamoylputrescine was prepared by the method of Smith and Garraway (1964). Further puri®cation of N-carbamoylputrescine by Amberlite CG-50 Na+ (type 2) column chromatography to remove N,N0 dicarbamoylputrescine and putrescine contamination was carried out as described by Akamatsu et al. (1978). N-Carbamoylputrescine (mp 185 C) was ®nally recrystallized from EtOH. Agmatine sulfate and arcaine sulfate were purchased from Sigma Chemicals. All other chemicals were of analytical reagent grade. Agmatineepoxy-Sepharose 6B was prepared by coupling agmatine sulfate (0.2 g) to epoxy-activated Sepharose 6B (7 g) in 50 ml of 0.1 M Na2CO3 at room temperature for 24 h. 3.2. Plant materials Seeds of maize (Zea mays L. cv. Goldencross Bantam T51) were soaked in water for 6 h at 25 C and then germinated in moist vermiculite in the dark or continuous light (ca 3000 lux, at plant level). The excised shoots were sterilized with 0.1% benzalkonium chloride for 10 min and thoroughly washed with deionized water. 3.3. Plant extracts
Fig. 2. SDS-PAGE of agmatine deiminase from maize shoots. Line 1, agmatine deiminase with standard proteins: myosin (200 kDa), bgalactosidase (116 kDa), bovine albumin (66.3 kDa), aldolase (42.4 kDa), and carbonic anhydrase (30 kDa); line 2, agmatine deiminase.
To examine the eect of dark and light on enzyme activity, the shoots (ca 5 g) grown in the dark and light were homogenized in a chilled mortar with 10 ml of 50 mM K Pi buer pH 6.5 containing 5 mM 2-mercaptoethanol, 1 mM EDTA, and 1 mM PMSF (buer A). The homogenate was centrifuged at 15,000 g for 20 min. The supernatant was dialyzed against 500 ml of 50 mM K Pi buer pH 6.5 containing 5 mM 2-mercaptoethanol and 0.1 mM EDTA (buer B), changed three times at
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H. Yanagisawa / Phytochemistry 56 (2001) 643±647
8±12 h intervals. The dialyzate was centrifuged at 15,000 g for 20 min. The supernatant was assayed for enzyme activity. 3.4. Enzyme assays Enzyme activity was assayed by measuring N-carbamoylputrescine with the diacetyl monoxime method of Boyde and Rahmatullah (1980). The standard assay mixture, consisting of 50 mmol of K Pi buer, pH 6.5, 2 mmol of agmatine sulfate, 1 mmol of sodium diethyldithiocarbamate (in the case of plant extracts and the enzymes puri®ed through steps 1±3 below) and enzyme solution in a total volume of 1 ml, was incubated for 30 min at 30 C. The reaction was initiated by adding the enzyme and terminated by the addition of 0.1 ml 50% TCA. After denatured protein was removed by centrifugation at 1500 g for 15 min (this treatment was omitted for the ®nal puri®ed enzyme), 3 ml of the chromogenic solution was added to 0.5 ml of the supernatant in a 10 ml screw tube. The samples were mixed vigorously and then boiled at 100 C for 5 min. After cooling to room temperature in the dark, the absorbance of the samples was measured at 530 nm. Protein was determined according to the method of Bensadoun and Weinstein (1976) with BSA as a standard. 3.5. Puri®cation Step 1. The shoots (ca 450 g) grown for 7 days in the dark were homogenized in a blender with 900 ml of buer A. The homogenate was squeezed through a nylon mesh (74 mm) and centrifuged at 15,000 g for 20 min. The supernatant was used for the next step. Some (ca 5 ml) of the supernatant was dialyzed as before and the enzyme activity was measured. Step 2. The supernatant from step 1 was stirred with DE52 equilibrated with buer B (supernatant/ DE52, 1 l/50 g) for 1 h, and then the DE52 was recovered in a Buchner funnel. After washing with 0.5 l of buer B, the DE52 was stirred again for 5min in 250 ml of buer B containing 30% ammonium sulfate saturation. The DE52 was removed in a Buchner funnel and re-washed with 50 ml of buer B containing 30% ammonium sulfate saturation. The ®ltrate was brought to 70% ammonium sulfate saturation and allowed to stand for 30 min. After centrifugation at 15,000 g for 20 min, the precipitate was dissolved with 30 ml of buer A and then dialyzed against buer B (three exchanges at 8±l2 h intervals). The dialyzate was centrifuged at 15,000 g for 20 min and the supernatant was used for the next step. Step 3. The supernatant from step 2 was applied to a DE52 column (2.515 cm) equilibrated with buer B. After washing with 200 ml of buer B, the enzyme was eluted with a linear gradient of 150 ml of buer B and
150 ml of 0.5 M KH2PO4. Active fractions were pooled and the pH was adjusted to 6.5 with 2 N KOH. Step 4. The enzyme solution from step 3 was concentrated with a Collodion Bag 13200E (Sartorius). The concentrated enzyme was passed through a Sephadex G-100 column equilibrated with buer B. Active fractions were pooled. Step 5. The pH of the result of step 4 was adjusted to 8.5 with 2 N KOH. The enzyme solution was applied to an agmatine-epoxy-Sepharose GB column (15 cm) equilibrated with 50 mM K Pi buer pH 8.5 containing 5 mM 2-mercaptoethanol and 0.1 mM EDTA (buer C). After washing with 50 ml of buer C and then 100 ml of 0.5 M K Pi buer pH 8.5 containing 5 mM 2mercaptoethanol and 0.1 mM EDTA (buer D), the enzyme was eluted by a pulse label method with 10 ml of buer D containing 10 mM agmatine sulfate. Active fractions were pooled and then dialyzed against 1 l of buer B changed three times at 8±12 h intervals. The dialyzate was used as the puri®ed enzyme and stored at 4 C. 3.6. Determination of molecular mass and SDS-PAGE The molecular mass of the enzyme was estimated by gel ®ltration with a Sephadex G- 100 column (1.280 cm) equilibrated with 50 mM K Pi buer pH 6.5 containing 0.1 M KCl and 5 mM 2-mercaptoethanol. The column was calibrated with BSA dimer (132 kDa), BSA (66 kDa), ovalbumin (45 kDa), and cyt. c (12.4 kDa) as standard proteins. SDS-PAGE was performed as described by Laemmli (1970). Proteins were visualized with a silver stain kit (Daiichi Pure Chemicals Co., Ltd). 3.7. Putrescine synthase and related enzyme activity The assay mixture of putrescine synthase was similar to that described by Srivenugopal and Adiga (1981). Two dierent amounts of the puri®ed enzyme (0.5 and 1 mg) were used. After incubation at 30 C for 2 h, the enzyme reaction was terminated by boiling for 10 min. After denatured protein was removed by centrifugation at 15,000 g for 20 min, the supernatant was concentrated with a rotary evaporator. Citrulline was determined by PC (iso-PrOH±pyridine±H2O±HOAc=8:8:4:1) with ninhydrin and p-dimethylaminobenzaldehyde reagents. The assay mixtures of ornithine transcarbamylase or putrescine transcarbamylase consisted of 50 mmol of K Pi buer, pH 7, 2 mmol of sodium carbamoyl phosphate and ornithine or putrescine, and enzyme (0.5 and 1 mg) in a total volume of 1 ml. After incubation at 30 C for 1 h, the enzyme reactions were terminated by the addition of 0.1 ml 50% TCA. After denatured protein was removed by centrifugation at 1500 g for 10 min, citrulline or N-carbamylputrescine in the supernatant (0.5 ml) was estimated with the standard assay method.
H. Yanagisawa / Phytochemistry 56 (2001) 643±647
References Akamatsu, N., Oguchi, M., Yajima, Y., Ohno, N., 1978. Formation of N-carbamyl putrescine from citrulline in Escherichia coli. Journal of Bacteriology 133, 409±410. Bensadoun, A., Weinstein, D., 1976. Assay of proteins in the presence of interfering materials. Analytical Biochemistry 70, 241±250. Boyde, T.R.C., Rahmatullah, M., 1980. Optimization of conditions for the colorimetric determination of citrulline, using diacetyl monoxime. Analytical Biochemistry 107, 424±431. Chaudhuri, M.M., Gosh, B., 1985. Agmatine deiminase in rice seedlings. Phytochemistry 24, 2433±2435. Cogoni, A., Pias, C., Farci, R., Melis, A., Floris, G., 1990. Hordeum vulgare seedlings amine oxidase. Puri®cation and properties. Plant Physiology 93, 818±821. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Park, K.H., Cho, Y.D., 1990. Puri®cation of monomeric agmatine iminohydrolase from soybean. Biochemical and Biophysical Research Communications 174, 32±36. Prasad, G.L., Adiga, P.R., 1986. Puri®cation and characterization of putrescine synthase from cucumber seedlings. A multifunctional enzyme involved in putrescine biosynthesis. Journal of Biosciences 10, 373±391.
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Smith, T.A., 1969. Agmatine iminohydrolase in maize. Phytochemistry 8, 2111±2117. Smith, T.A., 1990. Plant polyamine Ð metabolism and function. In: Flores, H.E., Arteca, R.N., Shannon, J.C. (Eds.), Polyamines and Ethylene: Biochemistry, Physiology and Interactions. American Society of Plants Physiologists, Rockville, MD, pp. 1±23. Smith, T.A., Garraway, J.L., 1964. N-Carbamylputrescine. An intermediate in the formation of putrescine by barley. Phytochemistry 3, 23±26. Srivenugopal, K.S., Adiga, P.R., 1981. Enzymic conversion of agmatine to putrescine in Lathyrus sativus seedlings. Journal of Biological Chemistry 256, 9532±9541. Suzuki, Y., Hirasawa, E., Yanagisawa, H., Matsuda, H., 1990. The enzymes of polyamine metabolism in higher plants. In: Flores, H.E., Arteca, R.N., Shannon, J.C. (Eds.), Polyamines and Ethylene: Biochemistry, Physiology and Interactions. American Society of Plants Physiologists, Rockville, MD, pp. 73±90. Suzuki, Y., Hagiwara, M., 1993. Puri®cation and characterization of diamine oxidase from Zea mays shoots. Phytochemistry 33, 995± 998. Yanagisawa, H., Suzuki, Y., 1981. Corn agmatine iminohydrolase puri®cation and properties. Plant Physiology 67, 697±700.
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Agmatine deiminase from maize shoots: puri®cation and properties Hiroshi Yanagisawa * Biology Department, Aichi University of Education, Igaya, Kariya, Aichi 448-8542, Japan Received 18 July 2000; received in revised form 8 November 2000
Abstract Agmatine deiminase was puri®ed to a speci®c activity of 537 nkat/mg protein using an improved procedure. The recovery was 47% and the enzyme was homogeneous and remarkably stable. The molecular mass of the enzyme as determined by gel ®ltration was 75 kDa, and SDS-PAGE suggests that the enzyme is a hetero climer composed of subunits of 43.5 and 44 kDa. The Km for agmatine was 12 mM and arcaine was shown to be a potent competitive inhibitor of the enzyme, with a Ki of 3.3 mM. The enzyme does not have either putrescine synthase activity or the activities of its components ornithine and putrescine transcarbamylase. These results distinctly demonstrate that agmatine deiminase is dierent from putrescine synthase. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Zea mays; Maize; Agmatine deiminase; Putrescine synthase; Enzyme puri®cation
1. Introduction Polyamines are synthesized from arginine and ornithine in higher plants (Smith, 1990). In the arginine pathway, agmatine deiminase (EC 3.5.3.12) hydrolyzes agmatine to N-carbamoylputrescine and ammonia (Smith, 1969). Agmatine deiminase has been puri®ed to homogeneity from maize shoots (Yanagisawa and Suzuki, 1981), rice (Chaudhuri and Gosh, 1985), and soybean (Park and Cho, 1990). On the other hand, putrescine synthase has been puri®ed to homogeneity from grass pea (Srivenugopal and Adiga, 1981) and cucumber (Prasad and Adiga, 1986). Putrescine synthase has activities similar to those of agmatine deiminase, putrescine transcarbamylase, ornithine transcarb amylase, and carbamate kinase in a single polypeptide, and converts agmatine and ornithine to ammonia, putrescine, and citrulline in the presence of inorganic phosphate. Prior to this study, little was known about the kinetics of agmatine deiminase, although it was reported in studies with soybean that this enzyme was a monomer similar to putrescine synthase. However, it was
* Tel.: +81-0566-26-2646; fax: +81-0566-26-2310. E-mail address: [email protected] edu.ac.jp
uncertain whether agmatine deiminase was putrescine synthase itself or clearly dierent from putrescine synthase. In this paper, the characteristics of agmatine deiminase puri®ed from maize shoots by an improved procedure were examined, and compared with those of agmatine deiminase and putrescine synthase from other plants. 2. Results and discussion 2.1. Agmatine deiminase assays Agmatine deiminase activity was previously determined by measuring the ammonia released from agmatine in Conway microdiusion units with Nessler reagent (Yanagisawa and Suzuki, 1981); however, this procedure is insensitive. Moreover, ammonia is released from agmatine by Cu-containing amine oxidase in higher plants, and the estimation of agmatine deiminase activity by measuring the ammonia released from agmatine with extracts including Cu-containing amine oxidase is not accurate. Cu-containing amine oxidase has been detected in several species of leguminous and dicotyledonous plants (Suzuki et al., 1990), and recently in monocotyledonous plants including maize (Cogoni et al., 1990; Suzuki and Hagiwara, 1993). Therefore,
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00491-X
644
H. Yanagisawa / Phytochemistry 56 (2001) 643±647
agmatine deiminase activity was determined by measuring N-carbamoylputrescine by the diacetyl monoxime method (Boyde and Rahmatullah, 1980). When measuring the enzyme activity of plant extracts and in an early puri®cation step that involved both agmatine deiminase and Cu-containing amine oxidase, 1 mM sodium diethyldithiocarbamate was added to the reaction mixture. This reagent is a potent inhibitor of Cu-containing amine oxidase and did not in¯uence either the enzyme activity or the colorimetric reaction. 2.2. Eect of dark and light on activities of agmatine deiminase from maize shoots Agmatine deiminase activities of maize shoots grown for 7 days in the dark and light at 25 C were 0.25 and 0.28 nkat/shoot, respectively. No signi®cant dierence was found between the enzyme activities of maize shoots grown in the dark and light for 7 days.
2.4. Kinetics of agmatine deiminase The enzyme reactions were linear with respect to time and enzyme concentrations under standard assay conditions. The optimal pH of the enzyme puri®ed by the improved procedure was 6.5, with 85% of the maximum activity at pH 6.0 and 8.0. The enzyme showed a Michaelis±Menten-type saturation curve. A Km for agmatine of 12 mM was obtained from double-reciprocal plots (Fig. 1). It has been reported that the Km values for agmatine of the enzymes previously puri®ed from maize shoots (Yanagisawa and Suzuki, 1981) and soybean axes (Park and Cho, 1990) were 0.19 and 5 mM, respectively. The high Km values reported previously might be due to an inaccurate estimation of the enzyme activity and the long reaction time. The dierence in the speci®city of agmatine against agmatine
2.3. Puri®cation of agmatine deiminase Agmatine deiminase from maize shoots grown for 7 days in the dark was puri®ed by a DE52 batchwise method, DE52 column chromatography, Sephadex G100 column chromatography, and agmatine-epoxySepharose 6B column chromatography. The puri®cation procedure was largely the same as that described previously (Yanagisawa and Suzuki, 1981), but with the following major modi®cations: 1 mM phenylmethyl sulfonyl ¯uoride (PMSF) and EDTA were added to the extraction buer; and the anity adsorbent was changed from agmatine±succinic acid-hexamethylenediamineSepharose 4B to agmatine-epoxy-Sepharose 6B. The enzyme was puri®ed to a speci®c activity of 537 nkat/mg protein, and obtained in 47% yield (Table 1). The enzyme was previously puri®ed to a speci®c activity of 230 nkat/mg protein and lost 30% of its original activity after 3 days at 4 C (Yanagisawa and Suzuki, 1981). However, the enzyme puri®ed with the improved procedure was stable for two months at 4 C without any loss of activity. The increase in speci®c activity and the outstanding stability of the enzyme suggests that the enzyme puri®ed with the improved procedure has not been modi®ed during puri®cation.
Fig. 1. Competitive inhibition of agmatine deiminase from maize shoots by arcaine. Control (closed circles), in the presence of 50 mM arcaine sulfate (open circles): 50 ng of the enzyme was used.
Table 1 Puri®cation of agmatine deiminase from maize shoots Puri®cation step
Total protein (mg)
Total activity (nkat)
Speci®c activity (nkat/mg)
Puri®cation (fold)
Yield (%)
1. Crude extract 2. Eluate from DE52 3. Eluate from DE52 column 4. Eluate from Sephadex G-100 column 5. Eluate from anity column
603 90.5 18.8 2.61 0.0944
107 92.9 87.1 68.2 50.7
0.178 1.03 4.63 26.2 537
1 5.75 26 147 3010
100 86.4 81 63.5 47.2
H. Yanagisawa / Phytochemistry 56 (2001) 643±647
deiminase and putrescine synthase could not be determined, since the Km for agmatine of putrescine synthase has not yet been reported. Arcaine, an analogue of agmatine, is reportedly a potent inhibitor of the enzyme (Smith, 1969; Yanagisawa and Suzuki, 1981), and this inhibition is competitive, with a Ki for arcaine of 3.3 mM (Fig. 1). It is unclear whether agmatine deiminase puri®ed from soybean and rice and putrescine synthase would also be inhibited by arcaine. 2.5. Subunit structure of agmatine deiminase The molecular mass of enzyme puri®ed by the improved procedure was determined to be 75 kDa by the gel ®ltration method with Sephadex G-100 column chromatography. SDS-PAGE of the enzyme showed very close polypeptides of 43.5 and 44 kDa (Fig. 2). Sugar moieties were not found in the enzyme as evidenced by the phenol sulfuric acid method. These results suggest that the enzyme is a hetero dimer, although those from maize (Yanagisawa and Suzuki, 1981) and rice (Chaudhuri and Gosh, 1985) were reported to be dimers, and that from soybean was reported to be a monomer (Park and Cho, 1990).
645
2.6. Putrescine synthase activity Putrescine synthase, puri®ed to homogeneity from grass pea (Srivenugopal and Adiga, 1981) and cucumber (Prasad and Adiga, 1986), exhibited agmatine deiminase activity of 0.39 and 0.73 nkat/mg protein, respectively. This enzyme was highly unstable: grass pea putrescine synthase lost its component activities within 48 h at 4 C after puri®cation. Agmatine deiminase puri®ed from maize did not have the activities of putrescine synthase (citrulline formation), putrescine transcarbamylase (Ncarbamoylputrescine formation), or ornithine transcarbamylase (citrulline formation). These results distinctly demonstrate that agmatine deiminase diers from putrescine synthase. 3. Experimental 3.1. Chemicals N-Carbamoylputrescine was prepared by the method of Smith and Garraway (1964). Further puri®cation of N-carbamoylputrescine by Amberlite CG-50 Na+ (type 2) column chromatography to remove N,N0 dicarbamoylputrescine and putrescine contamination was carried out as described by Akamatsu et al. (1978). N-Carbamoylputrescine (mp 185 C) was ®nally recrystallized from EtOH. Agmatine sulfate and arcaine sulfate were purchased from Sigma Chemicals. All other chemicals were of analytical reagent grade. Agmatineepoxy-Sepharose 6B was prepared by coupling agmatine sulfate (0.2 g) to epoxy-activated Sepharose 6B (7 g) in 50 ml of 0.1 M Na2CO3 at room temperature for 24 h. 3.2. Plant materials Seeds of maize (Zea mays L. cv. Goldencross Bantam T51) were soaked in water for 6 h at 25 C and then germinated in moist vermiculite in the dark or continuous light (ca 3000 lux, at plant level). The excised shoots were sterilized with 0.1% benzalkonium chloride for 10 min and thoroughly washed with deionized water. 3.3. Plant extracts
Fig. 2. SDS-PAGE of agmatine deiminase from maize shoots. Line 1, agmatine deiminase with standard proteins: myosin (200 kDa), bgalactosidase (116 kDa), bovine albumin (66.3 kDa), aldolase (42.4 kDa), and carbonic anhydrase (30 kDa); line 2, agmatine deiminase.
To examine the eect of dark and light on enzyme activity, the shoots (ca 5 g) grown in the dark and light were homogenized in a chilled mortar with 10 ml of 50 mM K Pi buer pH 6.5 containing 5 mM 2-mercaptoethanol, 1 mM EDTA, and 1 mM PMSF (buer A). The homogenate was centrifuged at 15,000 g for 20 min. The supernatant was dialyzed against 500 ml of 50 mM K Pi buer pH 6.5 containing 5 mM 2-mercaptoethanol and 0.1 mM EDTA (buer B), changed three times at
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8±12 h intervals. The dialyzate was centrifuged at 15,000 g for 20 min. The supernatant was assayed for enzyme activity. 3.4. Enzyme assays Enzyme activity was assayed by measuring N-carbamoylputrescine with the diacetyl monoxime method of Boyde and Rahmatullah (1980). The standard assay mixture, consisting of 50 mmol of K Pi buer, pH 6.5, 2 mmol of agmatine sulfate, 1 mmol of sodium diethyldithiocarbamate (in the case of plant extracts and the enzymes puri®ed through steps 1±3 below) and enzyme solution in a total volume of 1 ml, was incubated for 30 min at 30 C. The reaction was initiated by adding the enzyme and terminated by the addition of 0.1 ml 50% TCA. After denatured protein was removed by centrifugation at 1500 g for 15 min (this treatment was omitted for the ®nal puri®ed enzyme), 3 ml of the chromogenic solution was added to 0.5 ml of the supernatant in a 10 ml screw tube. The samples were mixed vigorously and then boiled at 100 C for 5 min. After cooling to room temperature in the dark, the absorbance of the samples was measured at 530 nm. Protein was determined according to the method of Bensadoun and Weinstein (1976) with BSA as a standard. 3.5. Puri®cation Step 1. The shoots (ca 450 g) grown for 7 days in the dark were homogenized in a blender with 900 ml of buer A. The homogenate was squeezed through a nylon mesh (74 mm) and centrifuged at 15,000 g for 20 min. The supernatant was used for the next step. Some (ca 5 ml) of the supernatant was dialyzed as before and the enzyme activity was measured. Step 2. The supernatant from step 1 was stirred with DE52 equilibrated with buer B (supernatant/ DE52, 1 l/50 g) for 1 h, and then the DE52 was recovered in a Buchner funnel. After washing with 0.5 l of buer B, the DE52 was stirred again for 5min in 250 ml of buer B containing 30% ammonium sulfate saturation. The DE52 was removed in a Buchner funnel and re-washed with 50 ml of buer B containing 30% ammonium sulfate saturation. The ®ltrate was brought to 70% ammonium sulfate saturation and allowed to stand for 30 min. After centrifugation at 15,000 g for 20 min, the precipitate was dissolved with 30 ml of buer A and then dialyzed against buer B (three exchanges at 8±l2 h intervals). The dialyzate was centrifuged at 15,000 g for 20 min and the supernatant was used for the next step. Step 3. The supernatant from step 2 was applied to a DE52 column (2.515 cm) equilibrated with buer B. After washing with 200 ml of buer B, the enzyme was eluted with a linear gradient of 150 ml of buer B and
150 ml of 0.5 M KH2PO4. Active fractions were pooled and the pH was adjusted to 6.5 with 2 N KOH. Step 4. The enzyme solution from step 3 was concentrated with a Collodion Bag 13200E (Sartorius). The concentrated enzyme was passed through a Sephadex G-100 column equilibrated with buer B. Active fractions were pooled. Step 5. The pH of the result of step 4 was adjusted to 8.5 with 2 N KOH. The enzyme solution was applied to an agmatine-epoxy-Sepharose GB column (15 cm) equilibrated with 50 mM K Pi buer pH 8.5 containing 5 mM 2-mercaptoethanol and 0.1 mM EDTA (buer C). After washing with 50 ml of buer C and then 100 ml of 0.5 M K Pi buer pH 8.5 containing 5 mM 2mercaptoethanol and 0.1 mM EDTA (buer D), the enzyme was eluted by a pulse label method with 10 ml of buer D containing 10 mM agmatine sulfate. Active fractions were pooled and then dialyzed against 1 l of buer B changed three times at 8±12 h intervals. The dialyzate was used as the puri®ed enzyme and stored at 4 C. 3.6. Determination of molecular mass and SDS-PAGE The molecular mass of the enzyme was estimated by gel ®ltration with a Sephadex G- 100 column (1.280 cm) equilibrated with 50 mM K Pi buer pH 6.5 containing 0.1 M KCl and 5 mM 2-mercaptoethanol. The column was calibrated with BSA dimer (132 kDa), BSA (66 kDa), ovalbumin (45 kDa), and cyt. c (12.4 kDa) as standard proteins. SDS-PAGE was performed as described by Laemmli (1970). Proteins were visualized with a silver stain kit (Daiichi Pure Chemicals Co., Ltd). 3.7. Putrescine synthase and related enzyme activity The assay mixture of putrescine synthase was similar to that described by Srivenugopal and Adiga (1981). Two dierent amounts of the puri®ed enzyme (0.5 and 1 mg) were used. After incubation at 30 C for 2 h, the enzyme reaction was terminated by boiling for 10 min. After denatured protein was removed by centrifugation at 15,000 g for 20 min, the supernatant was concentrated with a rotary evaporator. Citrulline was determined by PC (iso-PrOH±pyridine±H2O±HOAc=8:8:4:1) with ninhydrin and p-dimethylaminobenzaldehyde reagents. The assay mixtures of ornithine transcarbamylase or putrescine transcarbamylase consisted of 50 mmol of K Pi buer, pH 7, 2 mmol of sodium carbamoyl phosphate and ornithine or putrescine, and enzyme (0.5 and 1 mg) in a total volume of 1 ml. After incubation at 30 C for 1 h, the enzyme reactions were terminated by the addition of 0.1 ml 50% TCA. After denatured protein was removed by centrifugation at 1500 g for 10 min, citrulline or N-carbamylputrescine in the supernatant (0.5 ml) was estimated with the standard assay method.
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