Planta
Planta 142, 175-180 (1978)
9 by Springer-Verlag 1978
Succinic Semialdehyde Dehydrogenase of Wheat Grain L. Galleschi i, M.G. Tozzi 2, I. Cozzani 2, and C. Floris 1 Institute of Botany and 2Laboratory of Biochemistry, Faculty of Science, University of Pisa, Via Luca Ghini, 5. 1-56100 Pisa, Italy
Abstract. Succinic semialdehyde dehydrogenase (EC
1.2.1.16) was purified 74-fold from wheat grain (Triticure durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both N A D and N A D P support the oxidation of the substrate, but the former is 7-fold more active than the latter. The o p t i m u m p H for activity is around 9; the enzyme is stable in the p H range 6-9 and retains its whole activity up to 40 ~ C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K m for SSA and for N A D are 7.4x 10 . 6 M and 2 x 10 - 4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000. Key words: Aldehyde oxidation - Succinic semialdehyde dehydrogenase - Thiol compounds - Triticum.
Introduction
It has been reported that GABA, under aerobic conditions, is converted to succinic acid and oxidized through the Krebs cycle (Pietruszko and Fowden, 1961; Jakoby, 1962; Balfizs, et al., 1970; Chou and Splittstoesser, 1972). The metabolic pathway leading from G A B A to succinate through G A B A - T and S S A - D H was demonstrated in nervous tissue (Krnievi6, 1970; De Boer and Bruinvels, 1977), in plants (Dixon and Fowden, 1961 ; Streeter and Thompson, 1972) and in some microorganisms (Scott and Jakoby, 1959; J a k o b y and Abbreviations: GABA-7-aminobutyric acid; GABA-T-=7-aminobutyric acid transaminase ; ME = mercaptoethanol; SSA = succinic semialdehyde; SSA-DH = succinic semialdehydedehydrogenase
Scott, 1959; Kim and Tchen, 1962; Dover and Halpern, 1972) although the metabolic significance and the regulation mechanisms of this pathway, as well as the molecular properties of the enzymes involved, still await elucidation. The main source of G A B A (although not the unique) is the glutamate decarboxylation reaction, which is widespread in different tissues and microorganisms (Boecker and Snell, 1972; Cozzani, 1973). We described the properties of glutamate decarboxylase partially purified from Triticum durum grains (Galleschi et al., 1975), which is regulated by A T P and other nucleotides (Galleschi et al., 1976). A deeper understanding of the significance of the whole " G A B A - s h u n t " in wheat grains entails the characterization of the other two enzymes of the metabolic chain, namely, G A B A - T and SSA-DH. The partial purification and various properties of the latter enzyme from Triticum durum is described in this paper.
Material and Methods Enzyme Source
For a typical preparation, embryos from about 1000fruits caryopsis of I)'iticum durum Desf. (dry after-ripe, nondormant fruits, harvested in 1976 and stored at 10~ in the dark for 1 year) were used. The fruits were sterilized in 1% NaCI0 for 10 min, washed 6 times with deionized water, and dried in an air stream. The embryos were isolated from fruits by excision, as previously described (Meletti, 1959). Preparation and Partial Purification of SSA-DH
Unless otherwise stated, all the following operations were carried out at 4~ C. Extraction: To the isolated embryos, after v~eighing, the double
weight of alumina was added. Then the mixture was ground in
0032-0935/78/0175/$01.20
176 a mortar for about 20 min and suspended in 0.1 M potassium phosphate buffer, pH 7.35 containing 0.014 M ME. The mixture was stirred for 5 min and the insoluble material was then spun down by centrifugation at 3,500 g for 10rain.
Ammonium Sulfate Fractionation. The supernatant was brought to 40% saturation with ammonium sulfate and submitted to centrifugation at 10,000 g for 10 rain. The precipitate was discarded and the supernatant was brought to 60% saturation with ammonium sulfate. The centrifugation was then repeated under the same conditions and the precipitate was retained. Sephadex G 200 GelFiltration The precipitate from the ammonium sulfate fractionation was dissolved in 4 ml of 0.05 M potassium phosphate buffer, pH 7.35 containing 0.014 M ME, and applied to a Sephadex G 200 gel column (1.5 x 61 cm), equilibrated with the same buffer. Elution was performed with the same buffer at a flow rate of 15 ml/h, by collecting 3 ml fractions.
L. Galleschi et al. : Succinic Semialdehyde Dehydrogenase of Wheat Table 1. Purification of succinic semialdehyde dehydrogenase from Triticum durum Step
Total Total protein activity (mg) (Aabsorbance 340 nm/min)
Extraction
1114
19
1.7
100
40% (NH,)2SO~ precipitation
485
18
3.7
94
60% (NH4)2SO 4 precipitation
350
17.3
4.9
91
16.2
I26.0
85
Sephadex G-200 gel filtration
Storage. The active fractions from gel filtration (12 ml) were peeled, stored in stoppered tubes in the coid room, and used for the enzyme assays as described below. This preparation retains activity for about a week, in the presence of ME. Enzyme Assay SSA-DH activity was measured spectrophotometrically by following the reduction of NAD or NADP. The standard incubation mixture contained in a total volume of 1 ml:Tris-HCI buffer, pH 9.0 (80 gmol), NAD (l gmo[), ME (3 ~tmol), enzyme protein (about 15 gg). After 5 rain preincubation in a spectrophotometric cuvette thermostated at 30 ~ C, the reaction was started by addition of 0.5 gmol of succinic semialdehyde. In the control the substrate solution was replaced by an equal volume of water. The increase in optical density at 340 nm was recorded in a Saitron 30I spectrophotometer.
12.8
10- 2 x Specific Total activity yield (Aabsorbance (%) 340 nm/min/mg protein)
1110 _
9-
>
80_
E 60_
E ~'O
40.
O 0~
e~
20.
Protein Estimation The protein concentration of the enzyme preparations was measured by the Lowry method (Lowry etal., 1951).
pH Chemicals Succinic semialdehyde, NAD and NADP were supplied from Sigma Chemical Co., St. Lores, Mo., USA; Sephadex G-200 from Pharmacia Fine Chemicals, Uppsala, Sweden. The standard enzymes were obtained fiom Boehringer u.S. Mannheim, W. Germany. Alumina (aluminum oxide 90 standardized for chromatographic adsorption analysis according to Brockmann) was purchased from Merck A.G., Darmstadt, W. Germany. Other reagent and buffer salts were reagent grade materials.
Results
Purity and Specificity of the Enzyme A typical enzyme preparation, obtained by the extraction and purification procedure described above, is summarized in Table 1. The increase of specific activity during the gel-filtration steps could be due in part to removal of some physiologic inhibitors, e.g., Krebs'
Fig. 1. Dependence of SSA-DH activity on pH. 0.1 Tris-HCl buffer ( e - o ) ; 0.1 M gtycine-NaOH buffer (o-o)
cycle intermediates, affecting enzyme activity in the crude extracts (Galleschi, unpublished data). The following aldehydes at 1 mM concentration were not oxidized at appreciable rates : formaldehyde, glyoxylic acid, glutaraldehyde, benzaldehyde, anisaldehyde. At the same concentration, however, some of these compounds exerted considerable inhibition on the SSA-DH reaction, as reported below. Both pyridine coenzymes supported the oxidation of succinic semialdehyde. However, under standard assay conditions, the enzyme activity was reduced to 1/7 when NADP was used as a substitute for NAD. Effect of pH and Temperature The optimum pH for SSA-DH activity was centered around 9, with a sharp decrease in catalytic
L. Galleschi et al. : Succinic Semialdehyde Dehydrogenase of Wheat
177
x
% E
X
5.
=E
4.
E O
03
O
3 E
2~ e~
pH Fig. 2. Stability of SSA-DH at different pH values. Enzyme samples were stored for t h at 18 ~ C in 0.1 M Tris-acetate buffer either in the presence ( e - e ) or in the absence ( 9169 of 0.014 M ME. The residual enzyme activity was then determined under standard conditions
activity when the pH was shifted in either direction as shown in Figure I. The enzyme was stable in the neutral and alkaline pH range, without appreciable effect of mercaptoethanol (ME), losing activity at pH values under 6 (Fig. 2). The thermostability of the enzyme protein and the temperature-activity curve are also reported (Fig. 3).
Effect of Thiol Compounds The SSA-DH from Triticum durum was strongly activated by ME and became dependent on this activation
---tJ 2'5
3'o
3'5
4'0
temperature
4'5
5'o
~'~
6'o
(~
Fig. 3. Thermostability of SSA-DH and dependence of reaction velocity on the temperature. To check the thermostability ( o - o ) , samples of enzyme were kept 10 min at the temperature indicated, then quickly cooled in ice and the residual activity was measured under standard conditions. The dependence of reaction velocity on temperature ( o - o ) was measured in spectrophotometric cuvettes thermostated at the temperature values indicated
during the purification procedure. After exhaustive dialysis or G-200 gel filtration, the enzyme exhibited very low activity in the absence of ME. Addition of appropriate concentrations of this thiol compound restored enzyme activity. The dependence of reaction velocity on the ME concentration is shown in Figure 4. The preincubation time required for activation was about 1 min for ME in the tempera-
% • E
E Et -
5_
/
//
O
co 0 0 r-" _Q
e~
Planta 142, 175-180 (1978)
9 by Springer-Verlag 1978
Succinic Semialdehyde Dehydrogenase of Wheat Grain L. Galleschi i, M.G. Tozzi 2, I. Cozzani 2, and C. Floris 1 Institute of Botany and 2Laboratory of Biochemistry, Faculty of Science, University of Pisa, Via Luca Ghini, 5. 1-56100 Pisa, Italy
Abstract. Succinic semialdehyde dehydrogenase (EC
1.2.1.16) was purified 74-fold from wheat grain (Triticure durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both N A D and N A D P support the oxidation of the substrate, but the former is 7-fold more active than the latter. The o p t i m u m p H for activity is around 9; the enzyme is stable in the p H range 6-9 and retains its whole activity up to 40 ~ C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K m for SSA and for N A D are 7.4x 10 . 6 M and 2 x 10 - 4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000. Key words: Aldehyde oxidation - Succinic semialdehyde dehydrogenase - Thiol compounds - Triticum.
Introduction
It has been reported that GABA, under aerobic conditions, is converted to succinic acid and oxidized through the Krebs cycle (Pietruszko and Fowden, 1961; Jakoby, 1962; Balfizs, et al., 1970; Chou and Splittstoesser, 1972). The metabolic pathway leading from G A B A to succinate through G A B A - T and S S A - D H was demonstrated in nervous tissue (Krnievi6, 1970; De Boer and Bruinvels, 1977), in plants (Dixon and Fowden, 1961 ; Streeter and Thompson, 1972) and in some microorganisms (Scott and Jakoby, 1959; J a k o b y and Abbreviations: GABA-7-aminobutyric acid; GABA-T-=7-aminobutyric acid transaminase ; ME = mercaptoethanol; SSA = succinic semialdehyde; SSA-DH = succinic semialdehydedehydrogenase
Scott, 1959; Kim and Tchen, 1962; Dover and Halpern, 1972) although the metabolic significance and the regulation mechanisms of this pathway, as well as the molecular properties of the enzymes involved, still await elucidation. The main source of G A B A (although not the unique) is the glutamate decarboxylation reaction, which is widespread in different tissues and microorganisms (Boecker and Snell, 1972; Cozzani, 1973). We described the properties of glutamate decarboxylase partially purified from Triticum durum grains (Galleschi et al., 1975), which is regulated by A T P and other nucleotides (Galleschi et al., 1976). A deeper understanding of the significance of the whole " G A B A - s h u n t " in wheat grains entails the characterization of the other two enzymes of the metabolic chain, namely, G A B A - T and SSA-DH. The partial purification and various properties of the latter enzyme from Triticum durum is described in this paper.
Material and Methods Enzyme Source
For a typical preparation, embryos from about 1000fruits caryopsis of I)'iticum durum Desf. (dry after-ripe, nondormant fruits, harvested in 1976 and stored at 10~ in the dark for 1 year) were used. The fruits were sterilized in 1% NaCI0 for 10 min, washed 6 times with deionized water, and dried in an air stream. The embryos were isolated from fruits by excision, as previously described (Meletti, 1959). Preparation and Partial Purification of SSA-DH
Unless otherwise stated, all the following operations were carried out at 4~ C. Extraction: To the isolated embryos, after v~eighing, the double
weight of alumina was added. Then the mixture was ground in
0032-0935/78/0175/$01.20
176 a mortar for about 20 min and suspended in 0.1 M potassium phosphate buffer, pH 7.35 containing 0.014 M ME. The mixture was stirred for 5 min and the insoluble material was then spun down by centrifugation at 3,500 g for 10rain.
Ammonium Sulfate Fractionation. The supernatant was brought to 40% saturation with ammonium sulfate and submitted to centrifugation at 10,000 g for 10 rain. The precipitate was discarded and the supernatant was brought to 60% saturation with ammonium sulfate. The centrifugation was then repeated under the same conditions and the precipitate was retained. Sephadex G 200 GelFiltration The precipitate from the ammonium sulfate fractionation was dissolved in 4 ml of 0.05 M potassium phosphate buffer, pH 7.35 containing 0.014 M ME, and applied to a Sephadex G 200 gel column (1.5 x 61 cm), equilibrated with the same buffer. Elution was performed with the same buffer at a flow rate of 15 ml/h, by collecting 3 ml fractions.
L. Galleschi et al. : Succinic Semialdehyde Dehydrogenase of Wheat Table 1. Purification of succinic semialdehyde dehydrogenase from Triticum durum Step
Total Total protein activity (mg) (Aabsorbance 340 nm/min)
Extraction
1114
19
1.7
100
40% (NH,)2SO~ precipitation
485
18
3.7
94
60% (NH4)2SO 4 precipitation
350
17.3
4.9
91
16.2
I26.0
85
Sephadex G-200 gel filtration
Storage. The active fractions from gel filtration (12 ml) were peeled, stored in stoppered tubes in the coid room, and used for the enzyme assays as described below. This preparation retains activity for about a week, in the presence of ME. Enzyme Assay SSA-DH activity was measured spectrophotometrically by following the reduction of NAD or NADP. The standard incubation mixture contained in a total volume of 1 ml:Tris-HCI buffer, pH 9.0 (80 gmol), NAD (l gmo[), ME (3 ~tmol), enzyme protein (about 15 gg). After 5 rain preincubation in a spectrophotometric cuvette thermostated at 30 ~ C, the reaction was started by addition of 0.5 gmol of succinic semialdehyde. In the control the substrate solution was replaced by an equal volume of water. The increase in optical density at 340 nm was recorded in a Saitron 30I spectrophotometer.
12.8
10- 2 x Specific Total activity yield (Aabsorbance (%) 340 nm/min/mg protein)
1110 _
9-
>
80_
E 60_
E ~'O
40.
O 0~
e~
20.
Protein Estimation The protein concentration of the enzyme preparations was measured by the Lowry method (Lowry etal., 1951).
pH Chemicals Succinic semialdehyde, NAD and NADP were supplied from Sigma Chemical Co., St. Lores, Mo., USA; Sephadex G-200 from Pharmacia Fine Chemicals, Uppsala, Sweden. The standard enzymes were obtained fiom Boehringer u.S. Mannheim, W. Germany. Alumina (aluminum oxide 90 standardized for chromatographic adsorption analysis according to Brockmann) was purchased from Merck A.G., Darmstadt, W. Germany. Other reagent and buffer salts were reagent grade materials.
Results
Purity and Specificity of the Enzyme A typical enzyme preparation, obtained by the extraction and purification procedure described above, is summarized in Table 1. The increase of specific activity during the gel-filtration steps could be due in part to removal of some physiologic inhibitors, e.g., Krebs'
Fig. 1. Dependence of SSA-DH activity on pH. 0.1 Tris-HCl buffer ( e - o ) ; 0.1 M gtycine-NaOH buffer (o-o)
cycle intermediates, affecting enzyme activity in the crude extracts (Galleschi, unpublished data). The following aldehydes at 1 mM concentration were not oxidized at appreciable rates : formaldehyde, glyoxylic acid, glutaraldehyde, benzaldehyde, anisaldehyde. At the same concentration, however, some of these compounds exerted considerable inhibition on the SSA-DH reaction, as reported below. Both pyridine coenzymes supported the oxidation of succinic semialdehyde. However, under standard assay conditions, the enzyme activity was reduced to 1/7 when NADP was used as a substitute for NAD. Effect of pH and Temperature The optimum pH for SSA-DH activity was centered around 9, with a sharp decrease in catalytic
L. Galleschi et al. : Succinic Semialdehyde Dehydrogenase of Wheat
177
x
% E
X
5.
=E
4.
E O
03
O
3 E
2~ e~
pH Fig. 2. Stability of SSA-DH at different pH values. Enzyme samples were stored for t h at 18 ~ C in 0.1 M Tris-acetate buffer either in the presence ( e - e ) or in the absence ( 9169 of 0.014 M ME. The residual enzyme activity was then determined under standard conditions
activity when the pH was shifted in either direction as shown in Figure I. The enzyme was stable in the neutral and alkaline pH range, without appreciable effect of mercaptoethanol (ME), losing activity at pH values under 6 (Fig. 2). The thermostability of the enzyme protein and the temperature-activity curve are also reported (Fig. 3).
Effect of Thiol Compounds The SSA-DH from Triticum durum was strongly activated by ME and became dependent on this activation
---tJ 2'5
3'o
3'5
4'0
temperature
4'5
5'o
~'~
6'o
(~
Fig. 3. Thermostability of SSA-DH and dependence of reaction velocity on the temperature. To check the thermostability ( o - o ) , samples of enzyme were kept 10 min at the temperature indicated, then quickly cooled in ice and the residual activity was measured under standard conditions. The dependence of reaction velocity on temperature ( o - o ) was measured in spectrophotometric cuvettes thermostated at the temperature values indicated
during the purification procedure. After exhaustive dialysis or G-200 gel filtration, the enzyme exhibited very low activity in the absence of ME. Addition of appropriate concentrations of this thiol compound restored enzyme activity. The dependence of reaction velocity on the ME concentration is shown in Figure 4. The preincubation time required for activation was about 1 min for ME in the tempera-
% • E
E Et -
5_
/
//
O
co 0 0 r-" _Q
e~
