CC BY
25Russian Journal of Nematology, 2014, 22 (2), 131-140
Properties of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in third-stage larvae of the nematode Anisakis simplex -preliminary studies
Elzbieta Lopienska-Biernat1, Marta Czubak1, Ewa Anna Zaobidna1
and Jerzy Rokicki2
1 Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A,
10-917 Olsztyn, Poland
2 Invertebrate Zoology Division, University of Gdansk, al. Pilsudskiego 46, 81-378 Gdynia, Poland
e-mail: ela.lopienska@uwm.edu.pl
Accepted for publication 13 October 2014
Summary. The activities of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) were determined in third-stage larvae (L3) of Anisakis simplex. The optimum pH of TPS and TPP was 7.0 and optimum temperatures were 55 and 40°C, respectively. The thermostability of TPP was found to be higher than that of TPS. The activity of TPS at 20-50°C was approximately 20% of the maximum activity; at 65°C the enzyme was inactivated. The activity of TPP at 25-30°C was approximately 40% of the maximum activity; at 60°C the enzyme was inactivated. Effects of chemical compounds on the enzymes were determined. Supernatants used for enzyme preparations were obtained from homogenised worms spun for 15 min at 4°C and 1500 g. TPS activity increased up to 25-fold under the influence of trehalose. In the case of fructose and sorbitol an inverse relationship was shown between concentration and enzyme activity. Proline was demonstrated to be another TPS inhibitor. TPS was activated by 20 mM MgCl2, NaCl and KCl. On the other hand, it was inhibited by CuCl2, CaCl2 and CoCl2. TPP was activated by 10 mM MgCl2, CaCl2, CoCl2, ZnCl2 and NaCl and 20 mM FeCls, ZnCl, KCl and ethylenediaminetetra-acetic acid.
Key words: enzyme activity, pH, thermostability, third larval stage.
Anisakis simplex is a parasitic nematode that occurs mainly in fish (particularly cod, herring, sardines, sole and mackerel), as well as in dolphins and sea lions. In a human infected with A. simplex, larvae of the parasite stay alive in the body for a relatively short period, but the human host sometimes becomes highly sensitised. Hypersensitivity is particularly common in areas where there is a custom of eating raw, undercooked or improperly cooked fish (Danek & Rogala, 2005). The form of the parasite that invades humans is the third-stage larvae (L3), whose natural host is fish that live in salt or brackish waters. The larvae of this stage are extremely viable and resistant (Adams, 1999; Audicana et al., 2002; Molina-Garcia, 2002). Although this nematode is a serious epidemiological problem, there are still much data lacking on its metabolism, with metabolism of carbohydrates being particularly understudied. This is important
for endoparasites due to the oxygen-poor habitat in which they live (Lee & Atkinson, 1976). Trehalose is their main energy source (Behm, 1997). Studies on changes in this sugar add to knowledge of the metabolism of the nematode. Lopienska-Biernat et al. (2007) conducted research on the changes of carbohydrate in L3.
Understanding the metabolism of trehalose can provide an opportunity to enrich knowledge on the physiology and biochemistry of Anisakis and to develop treatments for diseases caused by these parasitic nematodes. Trehalose (a-D-glucopyranosyl-1-1-a-D-glucopyranoside) is a non-reducing disaccharide, composed of two glucose molecules with an unusual a-1,1-glycosidic bond. The bond combines the two molecules, eliminating the reducing properties. The disaccharide therefore has a high hydrophilicity and chemical stability. Hydrogen bonds formed between the hydroxyl
group of the sugar and phosphate of head groups of membrane phospholipids result in a very flexible glycoside bond (Wolska-Mitaszko, 2001), where several essential physiological functions are discharged, such as energy reserve, glucose uptake, and protection against stress (Solomon et al., 2000; Benoita et al., 2009). Trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) are the two key enzymes involved in trehalose synthesis. TPS catalyses the transfer of glucose from uridine diphosphate glucose (UDP)-glucose to glucose-6-phosphate to give trehalose-6-phosphate and UDP, while TPP removes phosphate to produce free trehalose (Behm, 1997; Elbein et al., 2003). In nematodes Dmitryjuk et al. (2012, 2013) characterised the properties of TPS and determined the activity of TPP in Ascaris suum and Kushwaha et al. (2011) determined the activity of TPP in Brugia malayi. Amongst nematodes, TPP was first identified in C. elegans where loss of its function caused lethal structural and metabolic defects in the nematode intestine (Kormish & McGhree, 2005), demonstrating its noteworthy significance in nematode species.
The aim of this study was to determine the optimum pH, optimum temperature, thermal stability and the impact of chemicals on the enzyme activity of TPS and TPP in L3 of A. simplex. Our results will help to better understand the physiology of the parasite and its response to various factors, which may be helpful in the development of appropriate drugs to combat anisakiasis.
MATERIALS AND METHODS
The effect of pH on the enzymes was determined in 0.1 M acetic acid-ammonia buffer within the range of pH 3.0-9.0.
Study of the optimum temperature of the enzymes was carried out in the temperature range 15-70°C in 0.1 M acetic acid-ammonia buffer at pH 7.0. For each subsequent sample, the temperature was increased by 5°C. To study the thermal stability of the enzyme, test samples containing buffered enzymatic protein were preincubated for 15 min at temperatures from 15-70°C. Next, the samples were cooled or warmed to 37°C and an enzymatic reaction was started by introduction of substrates: UDPG and G6P for TPS and T6P for TPP. Glucose, fructose, sorbitol, trehalose and proline were used as effectors for TPS activity. Sugars and amino acids were present in concentrations of 100, 200, 300 and 400 mmol. As effectors MgCl2, CaCl2, CuCl2, CoCl2, KCl2, NaCl2 were used with divalent ions present in concentrations of 2, 5, 10, 15, 20 and 25 mmol and monovalent ions at concentrations of 10, 20, 50, 100, 200, 400 mmol l-1. For assaying the influence of chemical compounds for activity of TPP 2, 5, 10 and 20 mmol l-1 solutions of the compounds MgCl2, CaCl2, CoCl2, ZnCl2, FeCl3, NaCl, KCl and ethylenediaminetetra-acetic acid (EDTA) were used. The activity of control sample, which contained water instead of the effector, was assumed at 100%.
Statistical analyses were conducted using the Statistica (Statsoft, V 9.0) software package. The data were analysed using the ANOVA, Post Hoc test; the results were considered statistically significant at P < 0.005.
The research material consisted of L3 of A. simplex from fresh Baltic herring (Clupea harengus). The fishes were acquired in the marketplace at Olsztyn. L3 were washed several times in 0.65% NaCl solution, then dried on filter paper. Samples (100 mg) of larvae homogenised in hand by a glass homogeniser, then followed by an electric homogenizer in 1:1 (w/v) ratio in TBS buffer. The homogenate was centrifuged at 1500 g for 15 min at 4°C.
The activity of TPS wad determined using a method described by Giaever et al. (1988) and activity of TPP by Kaasen et al. (1992). The end-product of the reaction (trehalose) was assayed by high-performance liquid chromatography using a method described by Dmitryjuk et al. (2009). The activity of TPS and TPP was expressed in unit (U) per mg protein measured with the Bradford (1976) method. One unit (U) defines the quantity of trehalose (^ mol) synthesis during 1 min at 37°C.
RESULTS
Effect of pH on the enzymes. The optimum pH for both enzymes was 7.0. Under acidic conditions at pH 3.0-4.5, TPS had no activity, and TPP had about 50% of the maximum activity. TPS and TPP activities were reduced to only 20% and 40%, respectively, at pH 7.5. At pH 8.5 an increase in activity of TPS to about 60% was observed; however, activity of TPP decreased to 20% of the maximum activity (Fig. 1).
Optimum temperature of enzyme activity. The activity of TPP was high (> 75%) at a relatively low temperature of 30°C. TPP showed optimal activity at 40°C. The activity of this enzyme decreased rapidly at a temperature higher than 50°C. By contrast, TPS became more active when the temperature rose above 45°C, its peak of activity being recorded at 55°C (Fig. 2).
Table 1. Effect of chemical compounds on the activity of trehalose-6-phosphate synthase (TPS) from third-stage larvae of A. simplex.
Chemical compounds Activity of enzyme [%]
[mmol l-1] 100 200 300 400
control 100.00
Glucose 54.5±16.44 34±5.02a 114.06±83.7 25.9±26.2
Fructose 234.5±212.9a 214.7±191a 36.7±10.42 -
Sorbitol 302.5±71.91a 95.8±90.2 9.11±1.48a 18.07±16.9a
Trehalose* 1578±463a 2376±276a 2494±111a -
Proline 21.15±11.6a 65.6±11.6a - -
The activities of the control sample without effectors (21.547 ± 2.23 U mg-1 of protein) were taken to be 100%; mean ± SD; n = 6; a - indicate significant difference means of the control and chemical groups. * - difference of control (inactivated enzyme preparation) and activated enzyme.
Table 2. Effect of divalent ions on the activity of trehalose-6-phosphate synthase (TPS) from third-stage larvae of A. simplex.
Chemical compounds Activity of enzymes [%]
[mmol l-1] 2 5 10 15 20 25
control 100
MgCl2 81.9±2.4 80.22±14.7 92.67±7.5 61.63±1.7a 135.05±0.9a 129.02±3.2a
CaCl2 32.53±6.5a 28.6±0.7a 81.12±2.6 59.9±5.7 85.08±7 90.27±10.1
CoCl2 50.79±21.1a 13.64±0.3a 33.2±3.9 36.69±0.4 49.46±16.9 43.96±0.9
CuCl2 60.86±36.7a 16.99±0.4a 20.83±11.8a 39.6±5.2 82.6±3.8 54.65±13.9
The activities of the control sample without effectors (21.547 ± 2.23 U mg 1 of protein TPS) were taken to be 100%; mean ± SD; n = 6; a - indicate significant difference means of the control and chemical groups.
|—*—TPP -"-TPC |
-- - J I
an l.D 15 511 55 EE E.5 1B 15 BE B5 9.D
PH
Fig. 1. Effect of pH on the activity of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) from third-stage larvae of Anisakis simplex. The activities of samples in pH 7.0 (0.41 ± 0.02 U mg-1 of protein for TPS) and (0.44 ± 0.02 U mg-1 of protein for TPP) were taken to be 100%. n = 9 (the number of independent enzyme preparations), error bars = standard deviation (SD).
120
0 -I-,-,-,-,-,-,-,-■-1-■-1-■-1-■-
20 25 30 35 40 45 50 55 60 65 70
tenperäbm [C]
Fig. 2. Effect of temperature on the activity of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) from third-stage larvae of Anisakis simplex. The activities of samples at 55°C (23.45 ± 2.12 U mg-1 of protein) and 40°C (0.375 ± 0.02 U mg-1 of protein) were taken to be 100%. n = 9 (the number of independent enzyme preparations), error bars = standard deviation (SD).
Fig. 3. Thermostability of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) from third-stage larvae of Anisakis simplex. The activities of samples at 60°C (15.34 ± 1.23 mg-1 of protein) and 40°C (0.17 ± 0.05 mg-1 of protein) were taken to be 100%. n = 9 (the number of independent enzyme preparations), error bars = standard deviation (SD).
Thermal stability of enzyme activity. The study was conducted in a temperature range of 20-70°C. TPS proved to be very thermolabile. TPS activity did not change over the 15 min pre-incubation period (without substrate) at temperatures of up to 50°C, with enzyme activity being in the range of 10-20% of the maximum activity. At 55°C TPS activity increased to about 40% of the maximum activity. At higher pre-incubation temperatures rising from 50 to 60°C there was an increase in the rate of enzyme activity. However, a pre-incubation temperature above 65°C resulted in the inactivation of the enzyme (Fig. 3). The standard deviations within a given temperature are small.
The thermal stability curve of TPP was different. An increase in pre-incubation temperature from 25
to 45°C was accompanied by increased activity of this enzyme. Temperature higher than 50°C resulted in inactivation of TPP.
Influence of carbohydrates and proline on activity of trehalose-6-phosphate synthase. Trehalose was the most potent activator of this enzyme at all tested concentrations. At a concentration of 300 mmol l-1 increase in the activity of TPS was up to 25-fold compared with the activity of the reference sample that did not contain the chemical compound. Also, fructose at concentrations of 100 and 200 mmol l-1 increased enzyme activity by more than 2-fold (Table 1). In the case of fructose and sorbitol, activity was found to be related to concentration. TPS was inhibited by
sorbitol at concentrations of 300 and 400 mmol l-1, having 9 and 18% of the activity of the reference sample, respectively. This chemical compound activated TPS by about 3-fold at concentration of 100 mmol l-1 (Table 1). Enzyme activity was inhibited to a lesser extent by proline at concentrations of 100 and 200 mmol l-1 having approximately 20 and 65% of its maximum activity. TPS was inhibited by glucose, with activity decreasing by about 70% at concentration of 200 mmol l-1 (Table 1).
Influence of chemical compounds. TPS. Of the divalent ions analysed, only Mg2+ ions were found to be activators of TPS. TPS activity increased by approximately 30% under the influence of Mg2+
ions at concentrations of 20 and 25 mmol l-1 (Table
2). It was observed that an inhibitory effect on TPS was exerted by heavy-metal ions, i.e., Cu2+ and Co2+. Cu2+ and Co2+ ions inhibited this enzyme activity by 83% even at a concentration of 5 mmol l-1 of salt. Also, Ca2+ ions at concentrations of 2 and 5 mmol l-1 caused inhibition of TPS activity by approximately 70% (Table 2).
TPP. The activity of TPP increased by 60% at the Mg2+concentration of only 10 mmol l-1 (Table
3). This enzyme activity was strongly stimulated by Co2+, Ca2+ and Zn2+ions at 10 mmol l-1. The presence of trivalent ions Fe in the reaction mixture elevated TPP activity by about 40% (Table 3).
Table 3. Effect of divalent and trivalent ions on the activity of trehalose-6-phosphate phosphatase (TPP) from third-
stage larvae of A. simplex.
Chemical compounds Activity of enzymes [%]
[mmol l-1] 2 5 10 20
control 100
MgCl2 59.2±4.5 81.7±12.2 158.6±10.8a 80.9±8.9
CaCl2 6.9±5.2 72.2±12.9 139±23.9 70.7±12.8
ZnCl2 140±3.8 76.4±12.4 150±12.8a 164±23.8a
CoCl2 63±12.5 84.5±12.4 139±12a 64.3±12.9
FeCl2 39.9±12.9 28±4.7a 79.6±23.9 137.7±23a
The activities of the control sample without effectors (0.257 ± 0.02 U mg 1 of protein TPP) were taken to be 100%; mean ± SD; n : 6; a - indicate significant difference means of the control and chemical groups.
Table 4. Effects of monovalent ions and ethylenediaminetetra-acetic acid (EDTA) on the activity of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) from third-stage larvae of A. simplex.
Chemical compounds Activity of enzymes [%]
[mmol l-1] 2 5 10 20 50 100 200 400
TPS
KCl - - 101.75±22 74.61±5.5 91.59±5.9 85.43±2.17 100.75±5.3 145.77±1.8a
NaCl - - 45.07±5.4 292.12±0.08a 466.65±50a 254.93±11.3a 33.76±8.2 27.07±1.9
TPP
KCl 131±12 80±12 50±34 124±12a - - - -
NaCl 174±23 126±23 204±54a 83±12 - - - -
EDTA 42±12.89 90±23.08 60±23.9 110±34.9a - - - -
The activities of the control sample without effectors (21.547 ± 2.23 U mg 1 of protein TPS; 0.257 ± 0.02 U mg 1 of protein TPP) were taken to be 100%; mean ± SD; n = 6; a - indicate significant difference means of the control and chemical groups.
Among the monovalent ions, Na+ ions were activators of TPS at concentrations of 20, 50 and 100 mmol l-1. Under the influence of Na+, enzyme activity was significantly increased, with 2- to 4fold higher activity as compared with the reference sample (Table 4). K+ ions did not affect the activity of this enzyme. Only K+ ions at the concentration of 400 mmol l-1 increased enzyme activity by approximately 45% compared with the activity of the reference sample (Table 4). Na+ ions increased TPP activity by 2-fold at the concentration of 10 mmol l-1; however, K+ ions activated this enzyme by 20% at the concentration of 20 mmol l-1. The activity of TPP was stimulated only by 10% with a 20 mmol l-1 concentration of EDTA (Table 4).
DISCUSSION
Carbohydrate metabolism plays a vital role in sustaining the processes of life in many nematodes, including A. simplex. The synthesis of trehalose during the development of parasites protects them from external factors so that they can survive in harsh conditions. The increase in the incidence of anisakiasis, due to the popular consumption of raw fish, has attracted the interest of researchers seeking new anti-parasitic drugs. Scientific evidence indicates that blocking the proteins involved in the enzymic synthesis of trehalose contributes to the death of nematodes, which can be used in developing measures to combat parasitic diseases (Honda et al., 2010). The first need is to know the properties of these proteins.
Both A. simplex enzymes examined (TPS and TPP) had optimum activity at pH 7. TPS was active at pH 5-9; however, TPP was active in the pH range of 3-9. The curves of TPS and TPP activity under different pH conditions are clearly marked with two maxima at pH 7.0 and 8.5 for TPS and pH 7.0 and 4.0 for TPP (Fig. 1), which may indicate the presence of isoforms of these enzymes. TPS from the muscle of the parasitic nematode A. suum shows the highest activity at acidic pH, with the optimum pH being 4.2. The enzyme exhibits at pH 7 is only about 29% of the maximum activity, and the enzyme in an alkaline environment is inactive (Dmitryjuk et al., 2013). TPS from Aphelenchus avenae and Saccharomyces cerevisiae is present in a complex with TPP, with optimum TPS activity at pH 8 and 8.5, respectively (Loomis et al., 2005; Chaudhuri et al., 2009). Also, according to the research of Pan et al. (2004) carried out in Mycobacterium smegmatis, the optimum activity of the enzyme is at pH 7. Trehalose synthase from Pseudomonas putida was examined and the enzyme
was found to have its highest activity at pH 7.4 (Ma et al., 2006). In the thermostable bacterium Thermus thermophilus RQ-1 synthase is an enzyme that is most active in a slightly acidic environment, having optimum activity at pH 6 (Silva et al., 2005). Valenzuela-Soto et al. (2004) investigated the properties of TPS and determined the environment for the optimum activity of the enzyme. The enzyme derived from Selaginella lepidophylla showed the highest activity at pH 7 and was stable within a pH range of 5-10. Similar to TPP of A. simplex, optimum pH was found to be 7.0 for A. suum (Dmitryjuk et al., 2012) and for B. malayi phosphatases (Kushwaha et al., 2011) and T. thermophilus (Silva et al., 2005). These data indicate that most of these enzymes require a neutral environment for proper activity.
The optimum temperature for TPS is 55°C, while that for TPP is 40°C. TPP activity depends on the temperature; as for optimum pH there seem to be two forms of the enzyme, which have optimum temperatures of 30 and 40°C. Similarly, TPP from muscle of A. suum and B. malayi and Escherichia coli has an optimum temperature of 35°C (Seo et al., 2000; Kushwaha et al., 2011; Dmitryjuk et al., 2012). In a study conducted by Dmitryjuk et al. (2013) TPS was inactive at 55°C; the temperature for optimum activity was 35°C. Also TPS from E. coli exhibits its highest activity at 35°C (Lee et al., 2005). Silva et al. (2005) showed that in T. thermophilus RQ-1 TPS-1 at 30°C is not activated, and the temperature for optimum activity is 98°C. At 102°C the enzyme has 80% of its maximum activity.
There are also differences in the thermal stability of enzymes in different species. TPS from A. simplex is the most stable at the temperature of 60°C. Dmitryjuk et al. (2013) showed that for enzymes of A. suum the highest thermal stability occurs at a temperature of 35°C. The difference in stability of TPS from A. simplex and A. suum may be indicative of the occurrence of enzyme in a complex with another protein that alters the properties of A. simplex TPS. Also, Ma et al. (2006) have demonstrated thermal stability of trehalose synthase of P. putida. Silva et al. (2005) studied the thermostable bacteria with a half-life of the enzyme at 100°C for 31 min. TPP from A. simplex represents a quite thermostable enzyme. Without the protection of substrate, the enzyme lost activity at just 50°C and had about 10% of its maximum activity. Similarly, TPP from B. malayi and A. suum retained significant activity at 50°C for 15 min. Other phosphatases were more thermostable. The phosphatases from Phormia regina showed 70-90%
of activity at 60°C. Also, TPP from E. coli retained 90% of its activity at 50°C (Seo et al., 2000). Mycobacterial and T. thermophilus RQ-1 enzymes also tolerated high temperatures (Klutts et al., 2003; Silva et al., 2005).
TPS activity increased under the influence of trehalose and fructose (Table 1). The enzyme derived from S. lepidophylla (Velenzuela-Soto et al., 2004) and from A. simplex increased their activity under the influence of fructose. Dmitryjuk et al. (2013), by examining the substrate specificity of the purified synthase of trehalose-6-phosphate from A. suum, showed that the enzyme is not specific with respect to fructose. However, the effect of trehalose had the opposite reaction on the enzyme. According to Velenzuela-Soto et al. (2004), trehalose did not affect the activity of the enzyme. The present work showed that TPS from A. simplex under the influence of trehalose increases its activity 25-fold, and is the most potent activator of the enzyme of the compounds tested (Table 1). Different results were also obtained when studying the effect of sorbitol on TPS activity. This compound accumulates in the cells under the influence of thermal and osmotic stress. Physiological concentrations of sorbitol protect the protein from heat, and thus from aggregation and inactivation. The accumulation of sorbitol under the influence of thermal stress is analogous to the accumulation of trehalose in yeast (Salvucci, 2000). TPS activity in A. simplex is increased 3-fold by sorbitol at a concentration of 100 mmol l-1, and then decreased with increasing concentrations (Table 1). The opposite effect of sorbitol on TPS activity of S. lepidophylla was demonstrated by Velenzuela-Soto et al. (2004). This compound is an activator of the enzyme. TPS activity increases with increasing concentration. At a concentration of 400 mmol l- , enzyme activity was 215% of relative activity. Proline accumulates in the cells of plants under osmotic stress, cooling, thermal stress, UV light and exposure to heavy metals (Ozturk & Demir, 2002). According to Claussen (2005), proline as a substance protects soluble complex protein structures against denaturation, stabilises membrane through interaction with phospholipids, and can be a source of energy and nitrogen. Proline reduces the oxidation of membranes and protects cells from reactive oxygen species (Hoque et al., 2007). High concentrations of proline can be harmful to plants (Ashraf & Foolad 2007). Research conducted by Velenzuela-Soto et al. (2004) confirms the inhibitory effect of proline on TPS activity from S. lepidophylla. Proline is also a TPS inhibitor in A. simplex (Table 1). In the present study it was found
that glucose is one of the substrates that has an inhibitory effect on TPS enzyme activity (Table 1). It can be concluded that accumulation of the enzyme substrate reduces enzyme activity. However, Velenzuela-Soto et al. (2004) showed that glucose activates TPS. The enzyme activity in S. lepidophylla increases with increasing concentration. Dmitryjuk et al. (2013) investigated the substrate specificity of the purified enzyme from A. suum and demonstrated that TPS is not specific for glucose.
The divalent ion Mg2+ was found to be an activator of TPS. The strongest activity of TPS was shown at the concentrations of 20 and 25 mmol l-1, with 130% relative activity (Table 2). Dmitryjuk et al. (2013) also showed that TPS activity from A. suum muscle under the influence of Mg2+ increased to approximately 160%. With another nematode, Aphelenchus avenae, Mg2+ ions were also an activator of the enzyme (Loomis et al., 2005). TPS from T. thermophilus RQ-1 also is activated by Mg2+ ions (Silva et al., 2005). In E. coli Mg2+ ions do not affect the activity of the enzyme, varying in the range of 95-107% (Lee et al., 2005). Velenzuela-Soto et al. (2004) found that in S. lepidophylla the activity of TPS is increased 3-fold. On the basis of the present study and other research to date it is clear that Mg2+ ions play an important role in TPP catalysis, as they do in nematodes (A. suum, B. malayi) and bacteria (M. smegmatis, T. thermophilus RQ-1) (Pan et al., 2004; Silva et al., 2005; Kushwaha et al., 2011; Dmitryjuk et al., 2012). It has been shown that the heavy-metal ions significantly decrease the activity of the enzyme (Table 3). This is related to the toxic properties of these compounds. Moreover, in P. putida (Ma et al., 2006), E. coli (Lee et al., 2005) and S. cerevisae (Chaudhuri et al., 2009) heavy-metal ions significantly decrease the activity of enzymes. Cu2+ ions at the concentration of 1 mmol l-1 will decrease enzyme activity by approximately 66% (Lee et al., 2005). Otherwise, Co2+ ions at 10 mmol/l activated TPP by about 40%, a similar effect having been found at 2 mmol l-1 only in thermophilic bacteria (Silva et al., 2005). Ca2+ ions affect TPS activity in many different ways. Concentrations of 2 and 5 mmol l-1 are inhibitors of the enzyme, whereas at higher concentrations activity is not altered (Table 2). Other results have been obtained by Dmitryjuk et al. (2013). These authors found that Ca2+ increases TPS activity of A. suum muscle by approximately 1.5-fold. The presence of Ca2+ ions is beneficial for enzyme activity in A. avenae (Loomis et al., 2005). Also Velenzuela-Soto et al. (2004) showed that the concentration of 1 mmol l-1 of Ca2+ increased the
enzyme activity in S. lepidophylla, but at the concentration of 10 mmol l- the enzyme was inactive. Lee et al. (2005) showed that low concentrations of Ca2+ do not affect the activity of TPS in E. coli; however, the concentration of 20 mmol l-1 inhibits its activity.
It was shown in the present study that Na+ is beneficial for TPS activity at concentrations of 50 and 100 mmol l-1, and TPP activity at 10 mmol l- . Similarly, a correlation was found for activity of TPP from A. suum; there Na+ ions increased activity about 3-fold and TPS activity by nearly 1.5-fold (Dmitryjuk et al., 2012, 2013). The activating effect of Na+ on the synthesis of trehalose-6-phosphate was also shown by Loomis et al. (2005) in A. avenae. Enzyme activity under the influence of Na+ ions was 2-fold. K+ ions increased the activity of the enzyme at the concentration of 400 mmol l- , while lower concentrations did not alter the activity (Table 4). A similar relationship was observed by Velenzuela-Soto et al. (2004) and Dmitryjuk et al. (2013), who showed that K+ ions are both an enzyme activator in S. lepidophylla as well as in the parasitic nematode A. suum. Also Loomis et al. (2005) have shown that K+ ions improve TPS activity in A. avenae 2-fold. However, TPP activity, unlike TPS, increased only at the highest tested concentration of 20 mmol l- . For TPP from A. suum K+ acts as an inhibitor, but at concentrations below 10 mmol (Dmitryjuk et al., 2012). A similar surprise was the activation of TPP by trivalent ion at 20 mmol l-1 by 40%, while in A. suum a concentration of 10 mmol l-1 produced an activation of 6-fold (Dmitryjuk et al., 2012).
The present study showed that the metal-chelating agent EDTA makes TPP a functionally stable enzyme at 20 mmol l-1 without change of activity. The results are different from those obtained in other nematodes. These differences may result from different concentrations of acid: the enzymes from A. suum and B. malayi were inhibited at 10 mmol l-1, while in the present study activity of the enzyme from A. simplex at 20 mmol l-1 did not change. Perhaps at a higher concentration EDTA ceases to be an inhibitor of TPP, especially, as it is a non specific metal chelator. Confirmation of differences in response to this effector in A. simplex L3 is the activation of trehalase, the enzyme responsible for the hydrolysis of trehalose (Lopienska-Biernat et al., 2007).
In conclusion, the available literature is concerned with the prevalence of forms of the enzymes TPS and TPP in nematodes, as a single protein or as protein complexes catalysing the reactions of trehalose synthesis. Both enzymes have
the potential to have isoenzymes. To further confirm this, the phenomenon is beginning to be studied at the molecular level. The present study has demonstrated significant differences in response to temperature and metal ions. It seems that the protein TPS is more sensitive than TPP.
REFERENCES
Adams, A.M., Miller, K.S., Welell, M.M. & Dong, F.M. 1999. Survival of Anisakis simplex in microwave-processed arrow tooth flounder
(Atheresthes stomias). Journal of Food Protection 62: 403-409.
Ashraf, M. & Foolad, M.R. 2007. Roles of glicine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59: 206-216.
Audicana, M.T., Ansotegui, I.J., Fernandez de Corres, L. & Kennedy, M.W. 2002. Anisakis simplex: dangerous-dead and live? Trends in Parasitology 18: 20-25. Behm, C.A. 1997. The role of trehalose in the physiology of nematodes. International Journal for Parasitology 27: 215-220.
Benoita, J.B., Martineza, G.L., Elnitsky, M.A., Lee, R.E. & Denlinger, D.L. 2009. Dehydration-induced cross tolerance of Belgica antarctica larvae to cold and heat is facilitated by trehalose accumulation. Comparative Biochemistry and Physiology (Part A: Molecular Integral Physiology) 152: 518-523. Chaudhuri, P., Basu, A., Sengupta, S., Lahiri, S., Dutta, T. & Ghosh, A.K. 2009. Studied on substrate specifity and activity regulating factor of trehalose-6-phosphate synthase of Saccharomyces cerevisiae. Biochimica et Biophysica Acta 1790: 368-374. Claussen, W. 2005. Proline as a measure of stress in
tomato plants. Plant Science 168: 241-248. Danek, K. & Rogala, B. 2005. [Anisakis simplex -hidden allergen of fish]. Alergia Astma Immunologia 10: 1-5 (in Polish). Dmitryjuk, M., Lopienska-Biernat, E. & Farjan, M. 2009. The level of sugar and synthesis of trehalose in Ascaris suum tissues. Journal of Helminthology 83: 237-243.
Dmitryjuk, M., Lopienska-Biernat, E. & Sawczuk, B. 2012. Properties of trehalose-6-phosphate phosphatase from Ascaris suum muscles -preliminary studies. Russian Journal of Nematology 20: 9-14.
Dmitryjuk, M., Dopieralska, M., Lopienska-Biernat, E. & Fr4czek, R.J. 2013. Purification and partial biochemical-genetic characterization of trehalose-6-phosphate synthase from adult female
Ascaris suum. Journal of Helminthology 87: 212-221.
Elbein, A.D., Pan, Y.T., Pastuszak, I. & Carroll, J.D. 2003. New insights on trehalose: a multifunctional molecule. Glycobiology 13: 17-27.
Giaever, H.M., Styrvold, O.B., Kaasen, I. & Ström, A.R. 1988. Biochemical and genetics characterization of osmoregulatory trehalose synthesis in Escherichia coli. Journal of Bacteriology 170: 2841-2849.
Honda, Y., Tanaka, M. & Honda, S. 2010. Trehalose extends longevity in the nematode Caenorhabditis elegans. Aging Cell 9: 558-569.
Hoque, M.D.A., Okuma, E., Banu, M.N.A., Nakumura, Y., Shimoishi, Y. & Murata, Y. 2007. Exogenus proline mitigates the detrimental effects of salt stress more than exogenus betaine by increasing antioxidant enzyme activities. Journal of Plant Physiology 164: 553-561.
Kaasen, I., Falcenberg, P., Stryvold, O.B. & STR0M, A.R. 1992. Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by KatF (AppR). Journal of Bacteriology 174: 889-898.
Klutts, S., Pastuszak, I., Edavana, V.K., Thampi, P., Pan, Y.T., Abraham, E.C., Carroll, J.D. & Elbein, A.D. 2003. Purification, cloning, expression and properties of mycobacterial trehalose-phosphate phosphatase. The Journal of Biological Chemistry 278: 2093-2100.
Kormish, J.D. & McGhee, J.D. 2005. The C. elegans lethal gut- obstructed gob-1 gene is trehalose-6-phosphate phosphatase. Developmental Biology 287: 35-47.
Kushwaha, S., Singh, P.K., Rana, A.K. & Misra-Bhattacharya, S. 2011. Cloning, expression, purification and kinetics of trehalose-6-phosphate phosphatase of filarial parasite Brugia malayi. Acta Tropica 119: 151-159.
Lee, D.L. & Atkinson, H.J. 1976. Physiology of Nematodes. UK, Macmillan Press Ltd. 360 pp.
Lee, J.H., Lee, K.H., Kim, C.G., Lee, S.Y., Kim, G.J., Park, Y.H. & Chung, S.O. 2005. Cloning and expression of a synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose. Applied Microbiology and Biotechnology 68: 213-219.
Loomis, S.H., Madin, K.A.C. & Crowe, J.H. 2005. Anhydrobiosis in nematodes: biosynthesis of trehalose.
Journal of Experimental Zoology 211: 311-320.
Lopienska-Biernat, E., Zöltowska, K. & Rokicki, J. 2007. Trehalose catabolism enzymes in L3 and L4 larvae of Anisakis simplex. Journal of Parasitology 93: 1291-1295.
Ma, Y., Xue, L. & Sun, D.-W. 2006. Characteristics of trehalose synthase from permeablized Pseudomonas putida cells and its application in converting maltose into trehalose. Journal of Food Engineering 77: 342-347.
Molina-Garcia, A.D. & Sanz, P.D. 2002. Anisakis simplex larvae killed by high- hydrostatic-pressure processing. Journal of Food Protection 65: 383-388.
Özturk, L. & Demir, Y. 2002. In vivo and in vitro protective role of praline. Plant Growth Regulation 38: 259-264.
Pan, Y.T., Edavana, V.K., Jourdian, W.J., Edmondson, R., Carroll, J.D., Pastuszak, I. & Elbein, A.D. 2004. Trehalose synthase of Mycobacterium smegmatis. Purification, cloning, expression, and properties of the enzyme. European Journal of Biochemistry 271: 4259-4269.
Salvucci, M.E. 2000. Sorbitol accumulation in whiteflies: evidence for a role in protecting proteins during heat stress. Journal of Thermal Biology 25: 353-361.
Seo, H.S., Koo, Y.J., Lim, Y.J., Song, J.T., Kim, C.H., Kim, J.K., Lee, J.S. & Choi, Y.D. 2000. Characterization of bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli. Applied and Environmental Microbiology 66: 2484-2490.
Silva, Z., Alarico, S. & Da Costa, M.S. 2005. Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties, of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. Extremophiles 9: 29-36.
Solomon, A., Salomon, R., Paperna, I. & Glazer, I. 2000. Desiccation stress of entomopathogenic nematodes induces the accumulation of a novel heat-stable protein. Parasitology 121: 409-416.
Valenzuela-Soto, E., Marquez-Escalante, J.A., Iturriaga, G. & Figueroa-Soto, C.G. 2004. Trehalose-6-phosphate synthase from Selaginella lepidophylla: purification and properties. Biochemical and Biophysical Research Communications 313: 314-319.
Wolska-Mitaszko, B. 2001. [Trehaloza-substancja przedziwna]. Wlasciwosci, wyst^powanie, zastosowania. Biotechnologia 2: 36-53 (in Polish).
E. Lopienska-Biernat, M. Czubak, E.A. Zaobidna and J. Rokicki. Особенности трегалоза-6-фосфат синтазы и трегалоза-6-фосфат фосфатазы у личинок 3-й стадии Anisakis simplex - предварительные наблюдения.
Резюме. У личинок 3-й стадии Anisakis simplex определяли активность ферментов трегалоза-6-фосфат синтазы (ТФС) и трегалоза-6-фосфат фосфатазы (ТФФ). Оптимум pH для ТФС и ТФФ составлял 7,0, а оптимум температур - 55°C и 40°C, соответственно. Было показано, что термостабильность ТФФ выше, чем у ТФС. Активность ТФС при температуре 20-50°C составляла около 20% максимальной активности, а при 65°C фермент инактивировался. Активность ТФФ при 25-30°C была около 40% максимальной активности, а инактивация наступала при 60°C. Определяли действие некоторых химических соединений на эти ферменты. Супернатанты для приготовления ферментов получали из гомогенизированных нематод после центрифугирования при 1500 g в течение 15 мин и температуре 4°C. Активность ТФС возрастала в 25 раз в присутствии трегалозы. Для фруктозы и сорбитола была показана обратная зависимость между концентрацией и активностью ферментов. Было показано, что пролин является еще одним ингибитором ТФС. ТФС активировалась при добавлении 20 mM MgCl2, NaCl и KCl. С другой стороны, этот фермент ингибировался CuCl2, CaCl2 и CoCl2. ТФФ активировался добавлением 10 mM MgCl2, CaCl2, CoCl2, ZnCl2 и NaCl, а также 20 mM FeCl3, ZnCl, KCl и этилендиаминтетрауксусной кислоты.
