Parasitology (1978), 76, 269-275 With 3 figures in the text
269
Activation of succinate dehydrogenase from adult Fasciola hepatica (Trematoda) J. BARRETT Department of Zoology, University College of Wales, Aberystwyth, By fed SY23 3DA (Received 22 September 1977) SUMMARY
The succinate dehydrogenase of adult Fasciola hepatica was found to exist in active and inactive forms. The enzyme was inactivated by 1 fiM oxaloacetate and activated by incubation with compounds which bind to the active site (succinate, fumarate, malonate) or by incubation with anions and certain nucleotides. The activation of the enzyme by succinate followed first-order kinetics. The extent of activation of F, hepatica succinate dehydrogenase depended on the nature and concentration of the activator and on the pH. The rate of activation of the enzyme depended on the temperature. In contrast, the fumarate reductase activity of F. hepatica was not activated by incubation with substrate or anions and was not inhibited by oxaloacetate (100 /im). The significance of these results in the regulation of the tricarboxylic acid cycle in parasitic helminths is discussed.
INTRODUCTION
It has long been known that the succinate dehydrogenase of mammals undergoes a relatively fast transformation from a low activity form to a high activity form when incubated with substrate, a process usually referred to as activation (Thorn, 1962). The active form of mammalian succinate dehydrogenase is deactivated by oxaloacetate, which is reversibly bound to the enzyme (Ackrell, Kearney & Mayr, 1974; Gutman, 1976). In addition to activation by succinate, the enzyme can be activated by incubation with compounds which bind to the active site (fumarate, malonate) and also by incubation with a variety of anions, nucleotides and reducing agents (for review see Singer, Kearney & Gutman, 1972). Parasites such as Fasciola hepatica have a highly modified tricarboxylic acid cycle and this may present unusual problems for metabolic control. In this paper the regulatory properties of the succinate dehydrogenase from F. hepatica are investigated and compared with succinate dehydrogenases from aerobic and anaerobic sources. MATERIALS AND METHODS
Adult F. hepatica were obtained from a local abattoir and brought to the laboratory in warm (37 °C) H6don Fleigs solution (Dawes, 1954). The whole worms were homogenized and the mitochondrial fraction prepared as described by
270
J. BARRETT
Van den Bossche (1972); the mitochondria were sedimented using an angle-head rotor operated for 20 min at 2 °C and 10000g (rav 10 cm). The final mitochondrial pellet was resuspended in 250 mil sucrose (pH 7-4) to give a concentration of approximately 5 mg protein/ml. The succinate dehydrogenase assay was modified from that of Metzger & Diiwel (1973). The reaction mixture contained (in 2 ml): MES buffer (2[2Vmorpholino]ethane sulphonic acid), pH 6-6, 50/tmol; 2,6-dichlorophenolindophenol, 0-05/imol; Na+ succinate, 100/unol; the decrease in extinction was followed a t 600 nm. The fumarate reductase assay was based on that of Prichard (1973); the reaction mixture contained (in 1 ml): Tris-HCl buffer, pH 7-2, 10 /tmol; MgCl2, 0-5/imol; NADH, 0-5fimol; CaCl2, 1-5/jmol; K+ fumarate, 5/imol; the assay was performed in an anaerobic cell and the decrease in extinction followed at 340 nm. The addition of ADP (2-5 mm final concentration) to the reaction mixture did not increase the activity of the enzyme. All assays were carried out at 25 °C using a Gilford Model 240 recording spectrophotorneter. The succinate dehydrogenase was inactivated by incubation with 2 /im. oxaloacetate and the effect of activators tested by incubation of the inactive enzyme with activator for 20 min at 30 °C in 25 mM MES buffer, pH 6-6. All nucleotides and cofactors were purchased from the Boehringer Corp. and were the highest purity available.
RESULTS
The succinate dehydrogenase of F. hepatica was found to be completely inactivated by incubation with 2 /*M oxaloacetate and inactivation was complete within 2 min at 20 °C. Unlike mammalian preparations (Thorn, 1962), the succinate dehydrogenase from F. hepatica was fully active when isolated and had first to be deactivated by incubation with 2 /IM oxaloacetate before the effects of possible activators could be studied. Incubation of the inactive enzyme with 100 mM succinate, 25 min fumarate or 10 mai malonate (final concentrations) for 20 min at 30 °C resulted hi 100% activation of the enzyme. The rate of activation of F. hepatica succinate dehydrogenase by substrate was temperature dependent (Pig. 1). The energy of activation (Ea) for the activation of F. hepatica succinate dehydrogenase by succinate was 10179 cal/mol (42600 joules/mol). For mammalian succinate dehydrogenase, the Ea for activation by succinate is in the range 31000-33000 cal/mol (Kearney, Ackrell, Mayr & Singer, 1974). The enzyme was routinely assayed at 25 °C; this represents a compromise, giving a reasonable rate of reaction and yet a sufficiently slow rate of activation by substrate (succinate) to allow initial rates of reaction to be measured. The activation of F. hepatica succinate dehydrogenase by substrate followed first-order kinetics (Fig. 2). In addition to activation by compounds which bind to the active site (succinate, fumarate, malonate) the succinate dehydrogenase of F. hepatica was also activated by a number of inorganic anions (Table 1). The extent of activation was dependent not only on the nature of the anion (Table 1),
Succinate dehydrogenase of F. hepatica
271
1-7
1-6
1-5
1-4
1-3
1-2
345 340 330 335 5 (1/r)xiO Fig. 1. An Arrhenius plot of the activation of succinate dehydrogenase from Fasdola hepatica by succinate (100 mM final concentration). The points are the means of 3 determinations. Ea = 42 600 J/mol. 320
325
1-0r
0-8
0-6
o 0-4
0-2
6
8 10 Time (min)
12
14
16
Fig. 2. The activation of succinate dehydrogenase from Fasciola hepatica by succinate (100 mM final concentration), showing first-order kinetics. The points are the means of 3 determinations. 18
PAR
76
272
J. BARRETT
i of succinate dehydrogenasefrom Fa Anion (100 mat final concentration; sodium salts)
Activation* (%) 66 63 59 55 53 48 40
Br-
NO 3 -
so 2 A c4Ici-
PO43-
* Enzyme deactivated with 2 /tai oxaloacetate; activated by incubation with anions for 20 min at 30 °C, pH 6-6. (Mean of 3 determinations.)
Table 2. The activation of succinate dehydrogenase from Fasciola hepatica by bromide ions Concentration of Br~ (mil, sodium salt)
Activation
10 50 100
(%) 27 34 42
* Enzyme deactivated with 2 /IM oxaloacetate; activated by incubation with bromide for 20 min at 30 °C, pH 6-6. (Mean of 3 determinations.)
but also on its concentration (Table 2). In addition, the activation of F. hepatica succinate dehydrogenase depended on the pH (Fig. 3). The pH curve is fairly flat between pH 5-5 and 6-5, and then drops sharply. The succinate dehydrogenase of F. hepatica was also activated by incubation with nucleotides (Table 3). A similar activation by ATP, ADP, ITP and IDP has been reported for mammalian succinate dehydrogenase (Kearney et al. 1974). It is, however, uncertain whether the activation is a specific nucleotide effect or is due to the anionic charge on the molecule. In contrast to succinate dehydrogenase, the fumarate reductase activity of F. hepatica preparations was not enhanced by pre-incubation with compounds which bind to the active site (fumarate, succinate, malonate), nor by incubation with anions. Incubation with oxaloacetate (10-100 fim) also did not inhibit the fumarate reductase activity of F. hepatica mitochondria. The succinate oxidation/fumarate reduction ratio for the F. hepatica enzyme was found to be 2-3.
DISCUSSION
Succinate dehydrogenase, isolated from different sources differs in its kinetic properties and these differences can be related to the metabolic needs of the tissue (Singer, Kearney & Kenney, 1973). The enzyme from aerobic sources such as mammals and plants has a high rate of succinate oxidation relative to fumarate reduction (for mammals the ratio is about 60). Succinate dehydrogenase from
Succinate dehydrogenase of F. hepatica 60
273
r
50 c o o
269
Activation of succinate dehydrogenase from adult Fasciola hepatica (Trematoda) J. BARRETT Department of Zoology, University College of Wales, Aberystwyth, By fed SY23 3DA (Received 22 September 1977) SUMMARY
The succinate dehydrogenase of adult Fasciola hepatica was found to exist in active and inactive forms. The enzyme was inactivated by 1 fiM oxaloacetate and activated by incubation with compounds which bind to the active site (succinate, fumarate, malonate) or by incubation with anions and certain nucleotides. The activation of the enzyme by succinate followed first-order kinetics. The extent of activation of F, hepatica succinate dehydrogenase depended on the nature and concentration of the activator and on the pH. The rate of activation of the enzyme depended on the temperature. In contrast, the fumarate reductase activity of F. hepatica was not activated by incubation with substrate or anions and was not inhibited by oxaloacetate (100 /im). The significance of these results in the regulation of the tricarboxylic acid cycle in parasitic helminths is discussed.
INTRODUCTION
It has long been known that the succinate dehydrogenase of mammals undergoes a relatively fast transformation from a low activity form to a high activity form when incubated with substrate, a process usually referred to as activation (Thorn, 1962). The active form of mammalian succinate dehydrogenase is deactivated by oxaloacetate, which is reversibly bound to the enzyme (Ackrell, Kearney & Mayr, 1974; Gutman, 1976). In addition to activation by succinate, the enzyme can be activated by incubation with compounds which bind to the active site (fumarate, malonate) and also by incubation with a variety of anions, nucleotides and reducing agents (for review see Singer, Kearney & Gutman, 1972). Parasites such as Fasciola hepatica have a highly modified tricarboxylic acid cycle and this may present unusual problems for metabolic control. In this paper the regulatory properties of the succinate dehydrogenase from F. hepatica are investigated and compared with succinate dehydrogenases from aerobic and anaerobic sources. MATERIALS AND METHODS
Adult F. hepatica were obtained from a local abattoir and brought to the laboratory in warm (37 °C) H6don Fleigs solution (Dawes, 1954). The whole worms were homogenized and the mitochondrial fraction prepared as described by
270
J. BARRETT
Van den Bossche (1972); the mitochondria were sedimented using an angle-head rotor operated for 20 min at 2 °C and 10000g (rav 10 cm). The final mitochondrial pellet was resuspended in 250 mil sucrose (pH 7-4) to give a concentration of approximately 5 mg protein/ml. The succinate dehydrogenase assay was modified from that of Metzger & Diiwel (1973). The reaction mixture contained (in 2 ml): MES buffer (2[2Vmorpholino]ethane sulphonic acid), pH 6-6, 50/tmol; 2,6-dichlorophenolindophenol, 0-05/imol; Na+ succinate, 100/unol; the decrease in extinction was followed a t 600 nm. The fumarate reductase assay was based on that of Prichard (1973); the reaction mixture contained (in 1 ml): Tris-HCl buffer, pH 7-2, 10 /tmol; MgCl2, 0-5/imol; NADH, 0-5fimol; CaCl2, 1-5/jmol; K+ fumarate, 5/imol; the assay was performed in an anaerobic cell and the decrease in extinction followed at 340 nm. The addition of ADP (2-5 mm final concentration) to the reaction mixture did not increase the activity of the enzyme. All assays were carried out at 25 °C using a Gilford Model 240 recording spectrophotorneter. The succinate dehydrogenase was inactivated by incubation with 2 /im. oxaloacetate and the effect of activators tested by incubation of the inactive enzyme with activator for 20 min at 30 °C in 25 mM MES buffer, pH 6-6. All nucleotides and cofactors were purchased from the Boehringer Corp. and were the highest purity available.
RESULTS
The succinate dehydrogenase of F. hepatica was found to be completely inactivated by incubation with 2 /*M oxaloacetate and inactivation was complete within 2 min at 20 °C. Unlike mammalian preparations (Thorn, 1962), the succinate dehydrogenase from F. hepatica was fully active when isolated and had first to be deactivated by incubation with 2 /IM oxaloacetate before the effects of possible activators could be studied. Incubation of the inactive enzyme with 100 mM succinate, 25 min fumarate or 10 mai malonate (final concentrations) for 20 min at 30 °C resulted hi 100% activation of the enzyme. The rate of activation of F. hepatica succinate dehydrogenase by substrate was temperature dependent (Pig. 1). The energy of activation (Ea) for the activation of F. hepatica succinate dehydrogenase by succinate was 10179 cal/mol (42600 joules/mol). For mammalian succinate dehydrogenase, the Ea for activation by succinate is in the range 31000-33000 cal/mol (Kearney, Ackrell, Mayr & Singer, 1974). The enzyme was routinely assayed at 25 °C; this represents a compromise, giving a reasonable rate of reaction and yet a sufficiently slow rate of activation by substrate (succinate) to allow initial rates of reaction to be measured. The activation of F. hepatica succinate dehydrogenase by substrate followed first-order kinetics (Fig. 2). In addition to activation by compounds which bind to the active site (succinate, fumarate, malonate) the succinate dehydrogenase of F. hepatica was also activated by a number of inorganic anions (Table 1). The extent of activation was dependent not only on the nature of the anion (Table 1),
Succinate dehydrogenase of F. hepatica
271
1-7
1-6
1-5
1-4
1-3
1-2
345 340 330 335 5 (1/r)xiO Fig. 1. An Arrhenius plot of the activation of succinate dehydrogenase from Fasdola hepatica by succinate (100 mM final concentration). The points are the means of 3 determinations. Ea = 42 600 J/mol. 320
325
1-0r
0-8
0-6
o 0-4
0-2
6
8 10 Time (min)
12
14
16
Fig. 2. The activation of succinate dehydrogenase from Fasciola hepatica by succinate (100 mM final concentration), showing first-order kinetics. The points are the means of 3 determinations. 18
PAR
76
272
J. BARRETT
i of succinate dehydrogenasefrom Fa Anion (100 mat final concentration; sodium salts)
Activation* (%) 66 63 59 55 53 48 40
Br-
NO 3 -
so 2 A c4Ici-
PO43-
* Enzyme deactivated with 2 /tai oxaloacetate; activated by incubation with anions for 20 min at 30 °C, pH 6-6. (Mean of 3 determinations.)
Table 2. The activation of succinate dehydrogenase from Fasciola hepatica by bromide ions Concentration of Br~ (mil, sodium salt)
Activation
10 50 100
(%) 27 34 42
* Enzyme deactivated with 2 /IM oxaloacetate; activated by incubation with bromide for 20 min at 30 °C, pH 6-6. (Mean of 3 determinations.)
but also on its concentration (Table 2). In addition, the activation of F. hepatica succinate dehydrogenase depended on the pH (Fig. 3). The pH curve is fairly flat between pH 5-5 and 6-5, and then drops sharply. The succinate dehydrogenase of F. hepatica was also activated by incubation with nucleotides (Table 3). A similar activation by ATP, ADP, ITP and IDP has been reported for mammalian succinate dehydrogenase (Kearney et al. 1974). It is, however, uncertain whether the activation is a specific nucleotide effect or is due to the anionic charge on the molecule. In contrast to succinate dehydrogenase, the fumarate reductase activity of F. hepatica preparations was not enhanced by pre-incubation with compounds which bind to the active site (fumarate, succinate, malonate), nor by incubation with anions. Incubation with oxaloacetate (10-100 fim) also did not inhibit the fumarate reductase activity of F. hepatica mitochondria. The succinate oxidation/fumarate reduction ratio for the F. hepatica enzyme was found to be 2-3.
DISCUSSION
Succinate dehydrogenase, isolated from different sources differs in its kinetic properties and these differences can be related to the metabolic needs of the tissue (Singer, Kearney & Kenney, 1973). The enzyme from aerobic sources such as mammals and plants has a high rate of succinate oxidation relative to fumarate reduction (for mammals the ratio is about 60). Succinate dehydrogenase from
Succinate dehydrogenase of F. hepatica 60
273
r
50 c o o
