Aspartate transaminase (AST) activity in the camel tick Hyalomma ... mass of AST II was 52 KDa for the native enzyme, composed of one subunit of 50 KDa. AST.
Experimental and Applied Acarology 25: 231–244, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii TAREK M. MOHAMED Molecular Biology Department, National Research Centre, Tahrir St., Dokki, Cairo, Egypt (Received 14 June 2000; accepted 13 February 2001)
Abstract. Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52 KDa for the native enzyme, composed of one subunit of 50 KDa. AST II had a Km value of 0.67 mM for α-ketoglutarate and 15.1 mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50◦ C for 15 min. The enzyme was activated by MnCl2 , and inhibited by CaCl2 , MgCl2 , NiCl2 , and ZnCl2 . Key words: camel, tick, Hyalomma dromedarii, embryogenesis, aspartate aminotransferase, purification, characterization
Introduction It is well known that ticks act as transmitting agents of numerous pathogens, including protozoa, bacteria, rickettsia and viruses infecting man, livestock, and wildlife. The tick borne diseases take a tremendous toll of health and wealth in the developed and developing regions of the world. Hyalomma dromedarii are ectoparasites on camels which are one of the main domestic animals in Egypt. The camel tick H. dromedarii transmits the virus of spring– summer encephalitis (Arthur, 1961). Aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1, aspartate aminotransferase, AST) is the best studied of aminotransferases. It has been purified from a wide range of bacteria (Kondo et al., 1984; Hayashi et al., 1993; Bartsch et al., 1996; Battchikova et al., 1996), fungi (Kagamiyama et al., 1984), protozoa (Lowe and Rowe, 1985, 1986), vertebrate (Martini et al., 1983; Lowe and Rowe, 1986; Vessal and Taher, 1995) and invertebrate (Lain-Guelbenzu et al., 1990). Hamed et al. (1990) reported that α-ketoglutarate, as one important intermediate in the Krebs cycle, carbohydrate and amino acid metabolism,
232 represented 44% of the accumulated organic acid in H. dromedarii during embryogenesis. As a result, we studied aspartate aminotransferase as one of the enzymes which are responsible for the metabolism of α-ketoglutarate. Studying tick embryogenesis on the molecular level will shed more light on the adaptability of tick cell as host and vector for different pathogens and will be helpful in identifying the factor regulating cell division and will be essential for the successful propagation of tick line. In this study AST was purified and characterized during embryogenesis as a biochemical tool for monitoring the tick during development. Materials and Methods Tick material Engorged Hyalomma dromedarii females were collected from camels in the market near Cairo and held at 28◦ C and 85% relative humidity condition. Eggs were collected daily from fertilized ovipositing female ticks and either frozen immediately and designated 0–1 day (−40◦ C) or incubated under the same conditions until the appropriate age transferred to frozen storage at intervals of three days (3, 6, 9 etc). The eggs frozen immediately were designated day 0. Chemicals Aspartic acid, α-ketoglutaric acid, sodium pyruvate, molecular weight markers for gel filtration, DEAE-cellulose for chromatography and all resins and reagents for electrophoresis were obtained from Sigma Chemical Co. Molecular weight markers for sodium dodecyl sulphate polyacrylamide gel electrophoresis and Sephacryl S-300 were obtained from Pharmacia. Other chemicals were of analytical grade. AST assay AST activity was determined by measurement of oxaloacetate produced during the assay according to the method of Bergmeyer and Bernt (1965). The samples were incubated at 37◦ C for 1 h in tubes containing 100 µM aspartic acid, 2 µM α-ketoglutaric acid, 20 mM sodium phosphate buffer, pH 7.5 and appropriate amount of enzyme to give a final volume of 1.0 ml. The reaction was stopped by the addition of 1 ml 2,4-dinitrophenylhydrazine reagent and allowed to incubate 20 min at room temperature and 10 ml 0.4 N sodium hydroxide was added. The absorbance was recorded at 505 nm. The enzymatically liberated oxaloacetate was calculated from a standard curve
233 using oxaloacetate (or pyruvate). One unit of enzyme activity was defined as the amount of enzyme producing 1 µmol oxaloacetate (pyruvate) per hour under the standard assay conditions. Purification of AST from 3-day-old H. dromedarii eggs Unless stated otherwise all steps were performed at 4–7◦ C using 20 mM sodium phosphate buffer, pH 7.5. Step 1: Preparation of crude extract Crude extract was prepared by homogenizing 5 g of 3-day-old eggs in 10 ml of 50 mM sodium phosphate buffer, pH 7.5, using a Teflon pestle homogenizer. The homogenate was centrifuged at 13, 200 × g for 20 min at 4◦ C to remove insoluble debris and the supernatant was designated as crude extract and dialyzed overnight against the same buffer. The dialyzate was then centrifuged at 16, 300 × g for 20 min to remove precipitated protein. Step 2: DEAE-cellulose chromatography The dialyzed supernatant from Step 1 was applied directly to a DEAEcellulose column (20 × 2.6 cm i.d.) equilibrated with 50 mM sodium phosphate buffer, pH 7.5. The adsorbed material was eluted with a stepwise gradient ranging from 0 to 0.4 M NaCl prepared in the same buffer at a flow rate of 60 ml/h and 10 ml fractions were collected. Protein fractions exhibiting AST activity were eluted with 0.0, 0.05, 0.1 and 0.2 M NaCl, respectively and designated AST I, II, III and IV, according to elution order. Step 3: Sephacryl chromatography AST II was applied to a Sephacryl S-300 column (95 × 1.6 cm i.d.) equilibrated with 50 mM sodium phosphate buffer, pH 7.5 and developed at a flow rate of 60 ml/h and 3 ml fractions were collected. The AST II was eluted with the same buffer. Protein determination Protein was determined either by measuring the absorbance at 280 nm (Warburg and Christian, 1942) or by the method of Bradford (1976) using bovine serum albumin as a standard. Buffers Buffers were prepared according to Gomori (1955), and the final pH was confirmed with a pH meter.
234 Polyacrylamide gel electrophoresis (PAGE) Electrophoresis under nondenaturing conditions was performed in 7.5% (w/v) acrylamide slab gel according to the method of Davis (1964) using a Trisglycine buffer, pH 8.3. Protein bands were located by staining with Coomassie Brilliant Blue R-250. Molecular weight determination Molecular weight was determined by gel filtration technique using Sephacryl S-300 (Andrews, 1965). The column (95 × 1.6 cm i.d) was calibrated with cytochrome C (12.4 KDa), carbonic anhydrase (29 KDa), bovine albumin (66 KDa), alchol dehydrogenase (150 KDa), catalase (240 KDa) and ferritin (440 KDa). Dextran blue (2,000,000) was used to determine the void volume (Vo ). Subunit molecular weight was estimated by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). SDS-denatured phosphorylase B (94 KDa), bovine serum albumin (67 KDa), ovalbumin (43 KDa), carbonic anhydrase (30 KDa) and trypsin inhibitor (20 KDa) were used as standards. Results Developmental changes The activities of AST in the eggs exhibited highest activity on day 3 (25.5 ± 1.2 units/g eggs) (P > 0.01), followed by a gradual decrease up to day 9 and then increased on day 12 (22 ± 0.7 units/g eggs) (P > 0.01), followed by a gradual decrease until day 24 (14 ± 0.5 units/g eggs) (Figure 1). Purification of AST from 3-day-old H. dromedarii eggs The purification of AST is summarized in Table 1. From the elution profile of the chromatography on DEAE-cellulose (Figure 2a), it can be seen that AST activity was detected in four peaks: the negative adsorbed fractions and the fractions eluted with 0.05, 0.1 and 0.2 M sodium chloride and designated as AST I, II, III and IV, respectively. Further purification was restricted to AST II. A Sephacryl S-300 column (Figure 2b) was used to obtain AST II with the highest possible specific activity (3.63 units/mg protein). Homogeneity The electrophoretic behaviour of the crude extract and sample from the final purification step of AST II are shown in Figure 3. One band was
235
Figure 1. Changes in AST activity during embryogenesis of H. dromedarii. Each point represents the mean of 3 runs for each developmental stage ± S.E.
Table 1. Purification scheme for H. dromedarii AST II Purification step
Total units∗
Total protein (mg)
Specific activity (units/mg protein)
Fold purification
Recovery %
Crude extract DEAE-cellulose 0.0 M NaCl (I) 0.05 M NaCl (II) 0.1 M NaCl (III) 0.2 M NaCl (IV)
51
228
0.12
–
100
10 26 46 75
0.4 0.63 0.25 0.173
3.3 5.25 2.1 1.44
3.63
30.25
Sephacryl S-300 AST II
4.0 16.4 11.5 13
13.8
3.8
7.8 32.1 22.5 25.5
27
∗ One unit of AST activity was defined as the amount of enzyme producing 1 µmol
oxaloacetate (pyruvate) per h under standard assay conditions.
236
Figure 2. (a) A typical elution profile for the chromatography of H. dromedarii AST on DEAE-cellulose column (20 × 2.6 cm i.d.) previously equilibrated with 20 mM sodium phosphate buffer, pH 7.5 at a flow rate of 60 ml/h and 10 ml fractions. (b) A typical elution profile for the chromatography of H. dromedarii AST II DEAE-cellulose fraction on Sephacryl S-300 column (95 × 1.6 cm i.d.) previously equilibrated with 20 mM sodium phosphate buffer, pH 7.5 at a flow rate of 60 ml/h and 3 ml fractions. Absorbance at 280 nm (•—•), AST activity (x- - - -x).
237
Figure 3. Polyacrylamide gel electrophoresis for H. dromedarii AST II during purification steps. 1-crude extract. 2-Sephacryl S-300 AST II.
detected on the gel after gel filtration on Sephacryl S-300 which indicated the homogeneity of the final preparation. Molecular weight determination of AST II AST II was applied to a Sephacryl S-300 column as previously described in the purification technique. The molecular weight of AST II was calculated from the calibration curve (Figure 4) and estimated to be 52,000 Da. This value was confirmed by SDS-PAGE under reducing condition (Figure 5). The enzyme was composed of one subunit with molecular mass of 50 KDa estimated from the calibration curve. Characterization of AST II Km values The Km values of AST II were estimated to be 0.67 mM for α-ketoglutaric acid (Figure 6a) and 15.1 mM for L-aspartic acid (Figure 6b). pH profile AST had pH optimum at sodium phosphate buffer, pH 7.5 (Figure 7a). Effect of temperature on stability The effect of temperature on the stability of AST II is shown in Figure 7b. The reaction mixture was pre-incubated at various temperature prior to substrate
238
Figure 4. Calibration curve for molecular weight determination of H. dromedarii AST II by Sephacryl S-300 column (95 × 1.6 cm i.d.).
Figure 5. SDS-PAGE under reducing condition for molecular weight determination of H. dromedarii AST II 1-Sephacryl S-300 AST II 2-Standard proteins.
239
Figure 6. Lineweaver burk plots of H. dromedarii AST II for (a) α-ketoglutarate, (b) L-asparate.
addition. The AST II enzyme was stable up to 50◦ C for followed by a strong decrease in activity between 50◦ C and 80◦ C. The enzyme loss 89% of its activity at 80◦ C. Effect of metal ions Table 2 shows the effect of different metal ions on the activity of AST II. While MnCl2 activated AST II with 118% activation, MgCl2 , NiCl2 , ZnCl2 and CaCl2 caused 77, 36, 26 and 52% of inhibition for AST II, respectively.
240
Figure 7. pH optimum (a) and effect of temperature on stability (b) of H. dromedarii AST II.
Discussion The AST activity of H. dromedarii during embryogenesis was the highest in 3-day-old eggs after which it decreased. The activity increased again in 12-day-old eggs, coinciding with increased α-ketoglutarate concentration in 12-day-old eggs (Hamed et al., 1990). As a result the change of AST during embryogenesis of H. dromedarii would have to be of greater physiological consequence. Using chromatography on DEAE-cellulose four forms of 3-day-old H. dromedarii eggs AST was demonstrated. One form of AST has been identified in Trichomonas vaginalis (Lowe and Rowe, 1985, 1986), Chalmydomonas reinhardtii (Lain-Gulebenzu et al., 1990) and Leishmania maxicana (Vernal et al., 1998). Some Trypanosoma spp. produce at least two isoenzymes (Kreutzer and Sousa, 1981) and in Toxoplasma gondii two
241 Table 2. Relative activity of H. dromedarii AST II toward different metal ions Metal ions
Concentration (mM)
% Relative activity AST II
CaCl2 MgCl2 MnCl2 NiCl2 ZnCl2
1.0 1.0 1.0 1.0 1.0
23 64 218 74 38
∗ The relative activity is the enzyme activity with metal ion compared with enzyme activity without added metal ions. Activity in absence of compounds was taken as 100%. The enzyme was preincubated for 15 min at 37◦ C with 1 mM metal ion prior to substrate addition. Each value represents the average of two experiments.
isoenzyme were described (Darde et al., 1988). Also, two peaks of AST were observed during DEAE-cellulose chromatography of protein extract from alkalophilic Bacillus (Battchikova et al., 1996). The native molecular mass of H. dromedarii AST II was 52 KDa. This value was confirmed by SDS-PAGE and represented a monomer with molecular weight 50 KDa. The native molecular weight of AST II was smaller than those for L. maxicana (90 KDa) (Vernal et al., 1998), T. vaginalis (100 KDa) (Lowe and Rowe, 1985; 1986) and C. reinhardtii (138 KDa) (LainGuelbenzu et al., 1990). The molecular weight of AST II was nearly similar to those for Arabidopsis thaliana (between 44 and 45 KDa) (Wilkie and Warren, 1998), and Bacillus stearotherophilus (40.5 KDa) (Bartsch et al., 1996). The Km values of H. dromedarii AST II for the α-ketoglutarate (0.67 mM) and L-aspartate (15.1 mM) were close to those for several other ASTs: C. reinhardtii (0.55 mM for α-ketoglutarate) (Lain-Guelbenzu et al., 1990), archaebacterium Haloferax mediterranei (0.75 and 12.6 mM for α-ketoglutarate and L-asparate, respectively) (Muriana et al., 1991). On the other hand, lower Km values of α-ketoglutarate and L-Asparatate were reported for T. vaginalis (0.044 and 0.96 mM, respectively) (Lowe and Rowe, 1985) and C. reinhardtii (0.55 and 2.53 mM, respectively) (Lain-Guelbenzu et al., 1990). Also, a low Km value of AST for thermophilic bacillus species of L-asparate (3 mM)
242 with a high Km value of α-ketoglutarate (2.6 mM) were detected (Sung et al., 1990). The pH optimum for H. dromedarii AST II was 7.5. This was consistent with the pH optimum for archaebacterium H. mediterranei with maximum pH ranging from 7.6 to 7.9 (Muriana et al., 1991). The optimum pH of AST for Bacillus Stearothermophilus was 8.0 (Bartsch et al., 1996), for L. mexicana 7.0 (Vernal et al., 1998), for human liver, 7.8 and for human mitochondrial isoenzyme 6.7 (Leung and Henderson, 1981). H. dromedarii AST II was stable up to 50◦ C and retained 59% of its activity after incubation at 70◦ C for 15 min. Also, AST from thermophilic bacillus species was most active at 70◦ C (Sung et al., 1990). AST from Thermus aquaticus has highly thermostable and lost 50% activity after incubation at 100◦ C for about 6 h (Walker and Wang, 1993). On the other hand, AST for thermoacidophilic archaebacterium Sulfolobus solfataricus has lost 50% of its activity when it was incubated at 100◦ C for 2 h (Marino et al., 1988).
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