ELSEVIER Biochimica et Biophysica Acta 1201 (1994) 466-472
BB. Biocht~Pic~a et Biophysica A~ta
A new S-adenosylmethionine decarboxylase from soybean axes
Yon Sik Choi, Young Dong Cho * Department of Biochemistry, College of Science, Yonsei University Seoul 120-749 Korea
Received 20 December 1993; revised 23 June 1994
Abstract
A new active S-adenosylmethionine decarboxylase (EC 4.1.1.50) (SAMDC II) was extracted from soybean (Glycine max) axes. The enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone) (MGBG)-Sepharose 6B chromatographies. The molecular weight of the native enzyme was 110 000, while the subunit molecular weights were 66 000 and 58000, indicating a heterodimeric structure. The K m value of the enzyme for S-adenosylmethionine was 16/xM, which is two times higher than that of previously reported S-adenosylmethionine decarboxylase (SAMDC I) (8.1 ~M). The specific activity of SAMDC II during the seed growth increased rapidly and reached its maximum on the second day after germination whereas that of SAMDC I reached its peak on the fourth day. MGBG was shown to inhibit SAMDC II competitively like SAMDC I. Carbonyl and sulfhydryl group specific reagents modified SAMDC II, resulting in the loss of enzymatic activity. Agmatine, the product of arginine decarboxylation catalyzed by arginine decarboxylase, inhibited the SAMDC II competitively ( K i = 40 # M ) while it inhibited the SAMDC II non-competitively ( K i = 600 mM). The possible role of the chronological appearance of SAMDC I! and SAMDC I, and properties of the enzyme are briefly discussed in connection with polyamine biosynthesis in soybean axes.
S-Adenosyl-L-methionine decarboxylase (EC 4.1.1.50), a key enzyme of polyamine biosynthesis, catalyzes the decarboxylation of S-adenosylmethionine (SAM) to yield S-adenosyl-(5')-3-methyl-thiopropylamine, which is the donor of the propylamine moiety for the biosynthesis of spermidine and spermine, respectively. In plants, the en- zyme has been found in carrot [1], Lathyrus sativus [2], oat [3], corn [4], potato [5], Chinese cabbage [6], tobacco callus [7], mungbean [8] and Vinca rosea [9], and has been thoroughly purified from Chinese cabbage and corn. We have recently purified and characterized the enzyme from
soybean (Glycine max) axes [10], the first reported to be inhibited by agmatine [10]. During the purification process, we found evidence for the presence of another S-adeno- sylmethionine decarboxylase. For convenience, we desig- nate the new enzyme as SAMDC II and the previously reported SAMDC as SAMDC I. In contrast to the monomeric SAMDC I, the purified SAMDC II was a heterodimer. To the best of our knowledge, a hetero-di- meric SAMDC has not been reported in plants, but has been found in E. coli [11] and mammalian cells [12]. The new SAMDC II is free of diamine oxidase, which is known to lead to an artificial decarboxylation of S-adeno- sylmethionine [2]. The activity of SAMDC II attained a maximum on the second day after germination, whereas that of SAMDC I reached its maximum on the fourth day. Furthermore, the new enzyme exhibits a much higher K m value, and severe inhibition by agmatine as compared to SAMDC I. The chronological appearance of the two SAMDCs with different g m values for the same substrate, and different degree of inhibition by agmatine, represents a novel mechanism by which polyamine biosynthesis may be regulated intracellularly.
Y.S. Choi, Y.D. Cho / Biochimica et Biophysica Acta 1201 (1994) 466-472 467
2. Materials and methods
2.1. Chemicals
S-Adenosyl[carboxy-14 C]methionine and S-adenosyl [methyl-3H]methionine were purchased from Amersham. Methylglyoxal-bis(guanylhydrazone) hydrochloride (MGBG) was purchased from Sigma. MGBG-Sepharose was prepared by the method of Markham et al. [13], except the coupling of MGBG to epoxy activated Sepharose (Sigma) was performed at pH 9. Decarboxylated SAM was kindly supplied by Dr. Kijiro Samejima, Faculty of Phar- macentical Sciences, Josai Univ. Keyakidai, Sakado, Saitama, Japan. Partisil 10 SCX (4.6 X 250 mm) HPLC column was purchased from Whatman. MDL73811 was kindly supplied by the Marion Merrell Dow Research Institute, France. All other reagents were obtained from Sigma or from Pharmacia Fine Chemicals.
1000 ml of the same buffer containing 100 mM NaCI, SAMDC II was eluted by the 500 ml linear gradient of 100 to 200 mM NaCI in the same buffer. Active fractions were pooled and concentrated by ultrafiltration (Amicon's stirred cell, YM-10). The concentrated SAMDC II was applied to the column of MGBG-Sepharose 6B, which had been equilibrated with a buffer containing 25 mM Tris-HC1, pH 7.6 and 15 mM 2-mercaptoethanol. The column was washed with the same buffer and then washed with the same buffer containing 1 M KC1. The enzyme was eluted with a buffer containing 50 mM potassium phosphate, pH 6.5, 15 mM 2-mercaptoethanol, 1 M KCI and 1 mM MGBG. Each eluted fraction was dialyzed overnight against the 25 mM Tris-HC1 (pH 7.6) buffer containing 15 mM 2-mercaptoethanol, and the active fractions were pooled.
2.5. Affinity chromatography
2.2. Plant materials
Soybean (Glycine max) seedlings were grown in plastic trays at 25°C in the dark. All plants were watered daily [14].
2.3. Enzyme assay
SAMDC II activity was assayed at 37°C for 60 min by the liberation of 14CO2 from S-adenosyl-[carboxy- 14C]methionine as substrate by the method of Yang and Cho [10]. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 nmol 14CO2 per h. Specific activity was expressed as units (U) /mg protein. SAM decarboxylase activity was a linear function of both incubation time and concentration under these conditions.
2.4. Purification of SAMDC H
Purification of the enzyme was carried out at 4°C. Two-day-old soybean axes (400 g) were blended in a chilled electric mixer with 500 ml of 25 mM Tris-HCl (pH 7.6) containing 15 mM 2-mercaptoethanol, 0.1 mM EDTA and 0.1 mM PMSF. The homogenate was filtered through four layers of gauze and clarified by centrifugation (13 000 x g, 30 min). The supernatant was adjusted to 30% satura- tion with solid (NH4)2SO 4 and stirred at 4°C for 5 h. The solution was then centrifuged and the pellet was discarded. The supernatant was brought to 50% saturation with solid (NH4)2SO 4 and treated as above, except that the pellet was retained. The pellet containing the enzyme was dis- solved with 30 ml of 25 mM Tris-HC1 (pH 7.6) containing 15 mM 2-mercaptoethanol and 0.1 mM EDTA. The col- umn was washed with the same buffer. The 500 ml linear gradient of 0 to 100 mM NaC1 in the equilibration buffer was used for the elution of SAMDC I. After washing with
The immunoaffinity column was prepared by coupling antibodies raised in rabbits against purified diamine oxi- dase from soybean axes to CNBr-activated Sepharose 4B according to the method described by Yang and Cho [10]. The immunoaffinity column (1 X 5 cm) was equilibrated with 1.5 mM phosphate buffer, pH 7.2 containing 2.7 mM KC1 and 0.14 M NaC1. The soybean extract was passed through this column to specifically absorb diamine oxi- dase. The resultant effluent showed no detectable diamine oxidase.
2.6. Analysis of decarboxylated SAM (dcSAM)
Analysis of dcSAM produced by the enzymatic reaction was based on isocratic HPLC separation using a cation-ex- change medium [15]. The purified enz~lme was incubated with 25 mM Tris-HC1 (pH 7.6), 15 mM 2-mercapto- ethanol, 0.1 mM EDTA, 10 /xM SAM at 37°C for 2 h in a total volume of 5 ml. The reaction was terminated by the addition of 2.5 ml of 1.5 M perchloric acid. After removal of the precipitate by centrifugation at 13000 x g for 10 min, the supernatant was applied to a column (1 cm X 10 cm) of Dowex-50 H ÷ in order to separate SAM and dcSAM from the contaminants of the incubation mixture. The resin is washed with 100 ml of 1 M HC1 and 200 ml of 2 M HC1, before the two sulfonium compounds are eluted with 6 M HC1. The eluate was evaporated to dryness under reduced pressure at 37°C and the residue dissolved in 0.2 ml of distilled water. HPLC analysis of dcSAM was done with Waters Model 660 liquid chro- matography. The solvent system consisted of 0.5 M am- monium formate (pH 4.0) run isocratically at a flow rate of 2 ml/min. The extract was eluted at room temperature through a Partisil 10 SCX column (4.6 mm X 250 mm) and detected at 254 nm. The produced dcSAM was quanti- tated by comparison of peak heights to those of known quantities of the authentic compound. For the recovery
468 Y.S. Choi, Y.D. Cho /Biochimica et Biophysica Acta 1201 (1994)466-472
test, the standard dcSAM was added to the reaction mix- ture in the absence of the enzyme, treated exactly as described above and the amount of dcSAM was deter- mined.
concurrently and the residual activity was calculated rela- tive to the appropriate control.
2.11. Specific labeling of the actice site of SAMDC H
2. 7. Protein Determination
Protein was determined by the Lowry procedure [16], with bovine serum albumin as the standard protein.
2.8. Determination of molecular weight
The molecular weight of the enzyme was estimated by gel filtration through a Sephacryl S-200 column (1.1 x 95 cm) according to the method of Andrew [17] and 5-20% polyacrylamide gradient gel electrophoresis. The column was calibrated with aldolase (158 000), bovine serum albu- min (66 000), ovalbumin (45 000) and myoglobin (17 200) as markers of known molecular weight. The gel molecular weight standards were thyroglobulin (670000), ferritin (440000), catalase (232000), lactate dehydrogenase (140 000) and albumin (67000).
2.9. Determination of subunit molecular weight.
Disc gel and SDS electrophoresis were carried out according to Laemmli [18]. Lysozyme (14300), trypsin inhibitor (20100), carbonic anhydrase (29 000), ovalbumin (45000), bovine serum albumin (66000) and phospho- rylase b (97000) served as molecular weight standard markers.
2.10. Carbonyl and sulfhydryl group modification
The purified SAMDC II and I were incubated with carbonyl group modification reagents such as NaBH 4, NaBH3CN, phenylhydrazine, hydroxylamine and semicar- bazide, and sulfhydryl group modification reagents such as NEM and DTNB for 20 min at 20°C. After incubation, the enzyme activity was assayed at 37°C for 60 min. In the case of the sulfhydryl group modification, 15 mM 2-mer- captoethanol was removed from the enzyme solution be- fore incubation. Control containing no reagents was run
Specific labeling was achieved by the modification of the method of Pegg et al. [19]. The specific labeling procedure used for the purified enzyme (20 /xg) consisted of incubation with 20 nM (220 000 dpm) [methyl-3 H]SAM, 200 mM NaBH3CN , 25 mM Tris (pH 7.6), 15 mM 2-mercaptoethanol, 0.1 mM EDTA at 37°C in a total volume of 0.5 ml. After 2 hr, 0.5 ml of 1.5 M perchloric acid was added and the pellet was collected by centrifuga- tion at 13000 X g for 10 min and washed twice by resus- pension in 1 ml of 1.5 M perchloric acid followed by sonication and centrifugation. The pellet was dissolved in distilled water and aliquots of the samples were mixed with an equal volume of 5% (w/v ) SDS, 5% 2-mercapto- ethanol, 10% glycerol and 62.5 mM Tris (pH 6.8). The mixture was heated at 100°C for 2 min and subjected to electrophoresis in 0.1% SDS-12.5% acrylamide gels. After electrophoresis, the protein bands corresponding to small and large subunits were sliced and treated with 30% H202 for 3 hr at 50°C in order to release the protein from gels. The released preparation was counted for radioactivity in 10 ml scintillation solution (667 ml toluene, 333 ml Triton X-100, 5.5 g PPO, 0.1 g POPOP).
2.12. Kinetic properties of MGBG:
Inhibition of purified SAMDC I and II were examined with various concentrations of SAM in the absence and presence of MGBG respectively. MGBG was added to the standard assay mixtures with various concentrations of SAM and the enzyme activities were assayed at 37°C for 60 rain. The data was plotted according to the Lineweaver-Burk mode.
3. Results and discussion
Purification and characterization of SAMDC I from 4 day-old soybean axes were reported earlier [10]. The pu-
Table 1 Purification of SAMDC II from the axes (400 g) of soybean (Glycine max)
Step Total protein Total activity Specific activity Recovery Purification (mg) (nmol/h) (nmol/h per mg) (%) (-fold)
a Assayed after dialysis and concentration. Two-day-old soybean axes were used. SAMDC activity was assayed at 37°C for 60 min by the liberation of 14CO2 from S-adenosyl-[carboxy- 14 C]methionine as substrate. One unit for enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 nmole 14CO2 per 1 h. Specific activity was expressed as U / m g protein.
Y.S. Choi, Y.D. Cho /Biochimica et Biophysica Acta 1201 (1994)466-472 469
rification procedures consisted of ammonium sulfate frac- tionation, DEAE-Sepharose and MGBG-Sepharose 6B chromatographies. During the purification, a very weak activity of another SAMDC was observed on the DEAE- Sepharose column. To determine whether another enzyme was present, the concentration of the linear gradient of NaC1 was changed from 0-100 mM [10] to 100-200 mM. After elution of SAMDC I on the DEAE-Sepharose col- umn by linear gradient (0-100 mM of NaC1) as described in Materials and Methods, the subsequent elution step is critical for the recovery of SAMDC II. A continuous gradient (0-200 mM) proved to be very ineffective in separating the two enzymes on the DEAE-Sepharose col- umn. Larger amounts of SAMDC II were obtained possi- bly due to the better separation from other proteins using a 100-200 mM linear gradient. Furthermore, the enzymatic activity of SAMDC II was tested from the beginning of germination; We homogenized the tissue samples taken each time, separated the two enzymes by DEAE-Sepharose column elution and pooled the active fractions of each enzyme. The specific activity of SAMDC I was observed to be maximal on the fourth day but the specific activity of SAMDC II was observed to reach a peak on the second day from germination. Therefore, we used two-day old soybean axes for the purification of the SAMDC II. The enzyme was purified 1222 fold with a 2.5% recovery from soybean (Table 1). The high purification fold of the en- zyme was achieved in the final step. Tests for diamine oxidase conformed its absence in the SAMDC II prepara- tion and putrescine was shown to be innocuous for SAMDC II. The preparation from Lathyrus sativus [2], however, contained an artificial putrescine-dependent activity which could be inhibited by catalase. This activity was attributed to the presence of a contaminating diamine oxidase in the SAMDC preparation which generated H20 2. SAMDC II from soybean, however, was free from diamine oxidase and putrescine independent. After the final step, no con- tamination could be detected with silver stained polyacryl- amide gel. The new enzyme was found to constitute 0.002 + 0.0005% of the soluble cell protein (mean of five experiments) and about 8 × 1 0 - 9 % of the wet cell weight.
To exclude the possibility that this enzymatic reaction is a distinct reaction or set of reactions resulting in the carboxyl release from SAM, analysis of another product, dcSAM, was performed. The produced dcSAM was identi- fied and separated from SAM by HPLC in a Partisil 10 SCX column (Fig. 1). Isocratic elution with 0.5 M ammo- nium formate has proved useful for checking. Retention times were extremely reproducible, provided the flow rate was kept constant and clean samples were injected, dc- SAM produced by the enzyme with authentic dcSAM and without it were eluted respectively and there always was only one peak which was increased in proportion to the amount of authentic dcSAM added. Under the same condi- tions the amount of released 14CO 2 and the produced dcSAM were measured from each separate reaction, re-
i SAM
J: IL
I
hi ,ll oc_
Fig. 1. Production of dcSAM from SAM by purified SAMDC If. The purified enzyme was incubated with 25 mM Tris-HCl (pit 7.6), 15 mM 2-mercaptoethanol, 0.1 mM EDTA, 10 /.~M SAM at 37~C for 2 h in a total volume of 5 ml. Reaction mixture was treated and HPLC analyzed as described in Section 2.
spectively. The extraction recovery of the standard dcSAM from the reaction mixture was about 80%. The stoichio- metric relationship between CO e and dcSAM produced by the enzyme was 1:0.8 under consideration of the recovery of dcSAM. From these results, we could conclude that the new enzyme, SAMDC II, catalyzed the decarboxylation of SAM to yield dcSAM and CO: in rate of 1:1.
As shown in Fig. 2, the specific activity of SAMDC I was observed to increase steadily, reaching a peak on the
100~ "E ~. 80 '
° t ~ 7o ~ 6°1
5o! '-~ 40
30 ,
t ,~ lO
/ \ \ \
i 2 3 4 s Plant Stage (days)
Fig. 2. Change in S-adenosylmethionine decarboxylases activities in soybean axes during seeding growth. SAMDC I ( • ) , SAMDC II (D). Soybean axes were collected at subsequent days and homogenized. The two enzymes were separated by salt gradient on a DEAE column according to the purification method. Total activities were assayed at 37°C by the liberation of 14CO2 from S-adenosyl[carboxy-14 C]methionine as substrate. Specific activity was expressed as pmol 14CO2/h per mg protein.
470 Y.S. Choi, Y.D. Cho / Biochimica et Biophysica Acta 1201 (1994) 466-472
Fig. 3. Determination of subunit molecular weight of the purified SAMDC II by SDS-polyacrylamide gel electrophoresis (12% acrylamide). A, phosphorylase b (97000); B, bovine serum albumin (66000); C, ovalbu- min (45 000); D, carbonic anhydrase (29 000); E, trypsin inhibitor (20100); F, lysozyme (14300).
Fig. 5. Determination of molecular weight of the purified SAMDC II by pore gradient gel electrophoresis. A, thyroglobulin (670000) : B, ferritin (440000); C, catalase (232000); D, lactate dehydrogenase (140000); E, albumin (67000).
fourth day after germination with subsequent decrease whereas the activity of SAMDC II was shown to increase sharply, reaching a peak on second day after germination. We previously reported that the soybean ornithine decar- boxylase activity peaked two days after germination [20] and the arginine decarboxylase activity peaked four days after germination [21]. At a very early stage in plant development, ornithine decarboxylase has been reported to play a vital role in polyamine biosynthesis [22], for which SAMDC is necessary to produce decarboxylated SAM. As far as the specific activity of SAMDC II is concerned, there is a perfect coupling of the enzymatic specific activi- ties between SAMDC II and ornithine decarboxylase in the soybean. After two days SAMDC I might fill in for SAMDC II, the specific activity of which is coupled with that of arginine decarboxylase.
The subunit molecular mass of SAMDC II was found by SDS-PAGE to be 66000 and 58000 (Fig. 3). The native molecular mass was determined to be 110000 by gel filtration on a Sephacryl S-200 column (Fig. 4) and 5-20% polyacrylamide gradient gel electrophoresis (Fig. 5), indicating hetero-dimeric structure. All SAMDCs pre- viously isolated in plants have monomeric structures, in-
5 .6
5.4 ~ \ ~ A ~-:_ SAMDC II ]
5.2 ~ ~t~ / MW=110,000
5 - ~ - . B
~ 4.8 "~'--. \
-~ 48 / ~....c 4.4i / .......
[ " ' - D 4.2J -~
0 0.1 0 . 2 0 . 3 0.4 0.5 0 . 6
Kav (Vo-Vo)/(Vt-Vo)
Fig. 4. Determination of molecular weight of the purified SAMDC 11 by calibrated Sephacryl S-200 gel filtration chromatography. A, aldolase (158000); B, bovine serum albumin (66000); C, ovalbumin (45 000); D, myoglobin (17200).
cluding the enzymes from soybean SAMDC I (66000), corn (25 000), tobacco callus (35 000) and Chinese cab- bage (35 000). But SAMDC II isolated from soybean was found to be a hetero-dimer in common with the enzyme from E. coli (19000 and 14000). This is the first report of nonidentical subunits in plant SAMDCs. In addition, the SAMDC II has a remarkably larger molecular weight when compared with any other plant SAMDCs.
SAMDC II was observed to obey typical Michaelis- Menten kinetics like SAMDC I as reported previously [10]. To confirm one substrate binding site and to exclude substrate stimulation or inhibition, Hill plots were con- structed. N value was turned out to be 1.1, confirming the presence of only one binding site. The K m value of the new enzyme was determined by the Lineweaver-Burk plot to be 16 /zM (Fig. 6). Other plots such as Hanes-Woolf and Eadie-Hofstee plots gave the same K m value. The K m value of SAMDC II for SAM (16 /zM) was almost two times as high as the value of SAMDC I (8.1 /zM) but lower than those of Chinese cabbage (38 /zM), E. coli (90 /~M), baker's yeast (90 #M) and rat liver (36 /xM). Compounds structurally related to SAM such as methylth- ioadenosine, S-adenosylhomocysteine and S-isobutyl-de- oxyadenosine turned out to be not attacked by SAMDC II (Table 2). Such trends were also shared by SAMDC I and
12
A 10 ._=
"5 E 6 e-
6 O 4 ,r
'1" 2-
0 -0.1
/ 7 14 Vmax = 0.72 CO*2nmole/min I
o11 • o13 o.s 1/ [SAM] (1/uM)
Fig. 6. Lineweaver-Burk plot of initial reaction velocities (nmole/min) for S-adenosylmethionine measured at various concentrations of S- adenosylmethionine.
ES. Choi, Y.D. Cho / Biochimica et Biophysica Acta 1201 (1994) 466-472 471
Table 2 Effect of substrate analogues and MDL73.811 (irreversible inhibitor) on soybean SAMDC II and I
Conc. (mM) SAMDC II SAMDC I
relative activity (%)
S-adenosylhomocysteine 0.5 98 102 1 96 109
S-iosbutyldeoxyadenosine 1 102 106 2 95 102
Methylthioadenosine 1 109.5 96 a 2 95.5 88 a
MDL73,811 1 (/xM) 3.5 8.7 10 (/~M) 3.2 5
a Data are from Yang and Cho [10] The purified SAMDC II and I were incubated with the analogues for 20 min at 20°C at the concentrations shown. After incubation, the enzyme activity was assayed and the resulting activity was expressed as percent- age of that in the absence of any compound.
other SAMDCs [6,21]. Not many suicide inhibitors for the enzyme responsible for synthesizing polyamine were re- ported. Suicide inhibitors are also known to inactivate an enzyme very specifically and irreversibly. According to the previous report [23-27], 5'-{[(Z)-4-amino-2-butenyl]meth- ylamino}-5'-deoxyadenosine (MDL73 811), an analogue of decarboxylated SAM, was verified to be a suicide inhibitor in animals and microorganisms. We have made an attempt to see if the inhibitor used in animals and microorganisms has an effect on the new enzyme, S A M D C II, and if so, to find a possible common property. The inhibitor was ob- served to inhibit the soybean S A M D C II completely (Ta- ble 2). Although the results were not comprehensive, the inhibitor could be useful to screen the enzymes from the different sources considering the inhibition by MDL73 811 in animals, microorganisms and plants.
The effects of various substances on the S A M D C II activity are summarized in Table 3. As shown in Table 3, Mg 2÷ has no effect on S A M D C II activity even at high concentrations (10 mM). The S A M D C activity from iso- lated mungbean, however, was significantly enhanced. Similarly to S A M D C I, S A M D C II was competit ively inhibited by MGBG, another potent inhibitor (Fig. 7) which also inhibited the enzymes from Chinese cabbage [6] and other species [28]. The K i value of S A M D C II was 0.23 /zM, which is smaller than the values of E. col i (20 ~ M ) and Chinese cabbage ( 0 . 6 / z M ) but larger than that of soybean S A M D C I (0.13 /xM).
SAMDCs are known to contain a covalently linked pyruvate instead of pyridoxal phosphate [29-31] which is known to be involved in the catalytic activity. Among all the ca rbony l g r o u p - m o d i f y i n g reagents , N a B H 4 , NaBH3CN, phenylhydrazine, hydroxylamine and semicar- bazide were used to probe the involvement of carbonyl groups in the catalytic function of S A M D C II (Table 3). NaBH3CN, an agent reducing Schiff base, e.g. between pyruvoyl group of the enzyme and substrate, inhibited the enzyme only in the presence of the substrate, SAM. Hy-
Table 3 Effects of magnesium, carbonyl and sulfhydryl group inhibitors on the activity of SAMDC II and I from soybean axes
Conc. (mM) SAMDC II SAMDC I
relative activity (%)
Mg 2+ 1 100 85 10 103 90
NaBH 4 1 90 92 10 68 73
NaBH 3 CN 1 82 63 10 52 56
Phenylhydrazine 1 69 86 10 25 31
Hydroxylamine 0.5 36 34 1 11 8
Semicarbazide 0.5 95 102 1 86 88
NEM 0.1 27 39 0.5 9 5
DTNB 0.1 73 49 0.5 35 32
a Data are from Yang and Cho [10]. The purified SAMDC II and I were incubated with magnesium, carbonyl and sulfhydryl group modifying agents for 20 rain at 20°C. After incubation, the enzyme activity was assayed and the activity found expressed as a percentage of that in the absence of any compound. When the sulfhydryl group inhibitors were tested, 2-mercaptoethanol was removed from the enzyme solution before incubation.
droxylamine was found to be the most effective inhibitor. The effects of these reagents were observed to vary and were in the order of hydroxylamine > phenylhydrazine > NaBH3CN > semicarbazide > NaBH 4. A similar behavior pattern of these reagents has been reported for corn en- zyme [2].
The specific labeling method [19] was used to deter- mine whether the carbonyl-group was present in the large (66000) or small subunit (58000). After electrophoresis, two protein bands corresponding to large and small sub- units were extracted from gels by treatment of 30% HeO: and counted for radioactivity. The radioactive protein band coincided with the location of the large subunit on SDS- PAGE and the other protein band corresponding to small
2000
1800 ~
"c 16001 .1= 1400~ ~ 1200
~ 1000 o ~ 8 0 0
~ 6 0 0
4O0
2OO
0 -0.1
KI = 0 .23 uM
J A , / / "
/ " /
/ /
/-// ...... /'/
0 0.1 0.2 0.3 0.4 1/[SAM] (1/uM)
Fig. 7. Inhibition of SAMDC II by MGBG as a function of SAM concentration. Without MGBG ([]). The concentrations of MGBG were 0.2 mM ( • ) and 0.4 mM (• ) .
472 Y..S. Choi, Y.D. Cho / Biochimica et Biophysica Acta 1201 (1994) 466-472
subunit exhibited no radioactivity at all. However, the radioactivity of the large subunit was low (500 c p m / 2 0 ~ g protein). For now it is not clear why the value is so low, but the low value might be due to several procedures including reduction of substrate-enzyme complex by NaBH3CN and washing to remove unreacted materials followed by electrophoresis. In addition, the yield or effi- ciency of each step is probably not high. A pyruvate-con- taining large subunit and a pyruvate-free small subunit of the active enzyme were also found for E. coli, a hetero-di- meric enzyme [11]. So, we suggested that SAMDC II has a similar subunit pattern to E. coli enzyme. Previous reports have shown that SAMDCs have sulfhydryl group involved in the catalytic activity [3,29]. Typical sulfhydryl group- modifying reagents such as NEM and DTNB also inhibited both soybean SAMDCs significantly (Table 3). NEM was observed to be an effective modifying reagent. The same inhibitory effect of NEM has been reported for corn [3] and E. coli [29]. From such cumulative results, we suggest that both SAMDC I and II have carbonyl and sulfhydryl groups for catalytic activity. But the location of the sulfhydryl group on different subunits in SAMDC II and the role of the small subunit should be clarified.
Little is known about the regulation of polyamine bio- synthesis in higher plants. For years, we have tried to find endogenous metabolites which could activate or inhibit enzymatic activity. We have shown earlier that SAMDC I has been inhibited by spermidine and spermine, respec- tively [10]. However, the concentration was so high that it seemed rather ineffective intracellularty. We have purified arginine decarboxylase from soybean and proven that argi- nine decarboxylase is inhibited by agmatine, a product of arginine decarboxylase [21]. Also, we have shown that SAMDC I was inhibited by the low concentration of agmatine and suggested that agmatine could be a novel regulator in soybean polyamine biosynthesis [10]. How- ever, SAMDC II was more sensitive toward agmatine, which inhibited the enzyme competit ively ( K i = 40 ~ M ) whereas SAMDC I was non-competit ively inhibited (Ki = 600 #M) . In responding to increasing accumulation of agmatine, SAMDC II could be first inhibited strongly and then SAMDC I could be inhibited, suggesting that dcSAM synthesis might be steadily curtailed and not prevent syn- thesis of intracellular spermidine and spermine completely. Therefore, both SAMDC I and II may play an important part in the regulation of polyamine biosynthesis by agma- tine during the earlier period of seeding growth since SAMDC II exhibits its activity before SAMDC I.
Acknowledgements
We are pleased to acknowledge the financial support of the Korea Science and Engineering Foundation (91050005).
We are grateful to Dr. Charles Danzin and the Marion Merrell Dow Research Institute for the supply of MDL73811. We are grateful to Dr. Keijiro Samejima for the supply of decarboxylated SAM.
mun. 181, 1181-1186. [11] Anton, D.L. and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822. [12] Pajunen, A., Croza, A., J~inne, O.A., Ihalainen, R., Laitinen, P.H.,
Stanley, B., Madhubala, R. and Pegg, A.E. (1988) J. Biol. Chem. 263, 17040-17049.
[13] Markham, G.D., Tabor, C.W. and Tabor, H. (1982) J. Biol. Chem. 257, 12063-12068.
[14] Kang, J.H. and Cho, Y.D. (1990) Plant Physiol. 93, 1230-1234. [15] Pegg, A.E., Poso, H., Shuttleworth, K. and Bennett, R.A. (1982)
Biochem. J. 202, 519. [16] Lowry, O.H., Rosebrough, J.J., Farr, A.L. and Randall, R.J. (1951)
J. Biol. Chem. 193, 265-275. [17] Andrew, P. (1964) Biochem. J. 91, 221-233. [18] Laemmli, U.K. (1970) Nature 227, 680-685 [19] Shirahata, A., Christman, K.L., Pegg, A.E. (1985) Biochemistry 24,
4417-4423. [20] Kim, D.C. and Cho, Y.D. (1993) Korean Biochem. J. 26, 192-197. [21] Park, Y.C. and Cho, Y.D. (1992) Korean Biochem. J. 25, 618-623. [22] Bagni, N., Barbirei, P. and Torriginai, P. (1983) Plant Growth
Regul. 2, 177. [23] Pegg, A.E. and Jacobs, G. (1983) Biochem. J. 213, 495-502. [24] Danzin, C., Marchal, P. and Casara, P. (1990) Biochem. Pharmacol.
