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Glutathione S-transferase in the insect Apis mellifera macedonica
Comparative Biochemistry and Physiology, Part C 139 (2004) 93 – 97 www.elsevier.com/locate/cbpc
Glutathione S-transferase in the insect Apis mellifera macedonica Kinetic characteristics and effect of stress on the expression of GST isoenzymes in the adult worker bee Athanasios I. Papadopoulosa,*, Irene Polemitoua, Pshychoula Laifia, Astero Yiangoua, Chrysoula Tananakib a
Laboratory of Animal Physiology, Department of Zoology, Faculty of Science, School of Biology, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece b Laboratory of Apiculture–Sericulture, Department of Agriculture, Geotechnical Sciences, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece
Received 2 June 2004; received in revised form 21 September 2004; accepted 22 September 2004
Abstract The glutathione S-transferase present in the adult worker bee Apis mellifera macedonica was purified and analyzed for its physicochemical and kinetic properties. The enzyme is heterodimeric with subunit molecular masses of 29 and 25 kDa, respectively. Two main isoenzymes with distinct kinetic properties are present, with isoelectric points of 7.40 for the alkaline and 4.58 for the acidic forms, respectively. The two enzymes are induced independently by factors such as insecticide treatments and environmental conditions, including low temperatures or starvation. D 2004 Elsevier Inc. All rights reserved. Keywords: Glutathione S-transferase; Adult worker bee; Apis mellifera macedonica; Detoxification; Isoenzymes; Induction
1. Introduction Glutathione S-transferases (GSTs) are present in almost all animals and in most of them in multiple isoenzymic forms, constituting a significant intracellular mechanism of detoxification. The enzymes catalyse the conjugation of a large variety of compounds bearing an electrophilic site, with reduced glutathione (GSH) (Mannervik and Danielson, 1988). In insects, they represent a very interesting detoxification mechanism due to their involvement in tolerance to insecticides (Motoyama and Dauterman, 1980; Clark et al., 1985; Fournier et al., 1992; Kostaropoulos et al., 2001). Several authors have noted that increases in both GSH content and GST activity are
* Corresponding author. Tel.: +30 2310 998359; fax: +30 2310 998269. E-mail address: [email protected] (A.I. Papadopoulos). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.09.010
associated with resistance to organophosphorus insecticides (Reidy et al., 1990; Clark, 1989). The multiple isoenzymic forms of GST are distinguished by differences in structure and catalytic properties. The expression of isoenzymes depends on many internal and external factors. Age-dependent alteration of GST activities has been demonstrated in both vertebrates and invertebrates (Gregus et al., 1985; Hazelton and Lang, 1983; Kostaropoulos et al., 1996). Alteration of GST expression induced by various substances in insets has also been reported (e.g., food quality and administration of certain insecticides) (Hayaoka and Gauterman, 1982; Papadopoulos et al., 1999; Papadopoulos et al., 2000; Kostaropoulos et al., 2001). Significant alterations in the isoenzymic profile, leading to the suggestion that the multiple GST isoenzymes present in insects are regulated independently, have also been reported as a result of stress, caused by a variety of factors (Papadopoulos et al., 1999, 2001).
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In our previous work (Papadopoulos et al., 2004), we detailed alterations in isoenzymic pattern during the development of the honeybee, Apis mellifera macedonica. In the present paper, we report on the purification of GST from the adult worker bee, its kinetic characteristics, its molecular mass, the isolation of isoenzymes from purified enzyme, and their kinetic characteristics. We also report on the effect of environmental parameters upon the expression of the isoenzymic profile.
activity that remained bound to the column was eluted with high salt (3 M NaCl in buffer A, designated as buffer C). The purification procedure was carried out at 4 8C. The protein fractions eluted by buffers B and C were pooled and concentrated twice against 10 mM potassium phosphate buffer at pH 6.5 in an Amicon ultrafiltration cell, and the concentrate was either subjected to isoelectric focusing (IEF) or chromatofocusing at a pH range 7–4, as recommended (Pharmacia) on a 0.36-cm column. 2.4. Substrate specificity
2. Materials and methods 2.1. Chemicals and reagents The reagents for chromatofocusing, epoxy-activated Sepharose 6B, and the low-molecular-weight and broad pI calibration markers were purchased from Pharmacia (Uppsala, Sweden). All other chemicals were from Sigma (St. Louis, MO, USA). 2.2. Insects Honeybees (A. mellifera macedonica) were supplied from the apiary of the laboratory Apiculture in Aristotle University of Thessaloniki. 2.3. Preparation of cytosolic fractions and purification of the enzyme The cytosolic fraction was prepared from whole bodies of 10–15 individuals, as described previously (Kostaropoulos et al., 1996), using a modified homogenization medium (10 mM potassium phosphate buffer, pH 7.4, 0.25 M sucrose, 0.2 mM EDTA, 10 mM 2-mercaptoethanol (ME), and 25 AM PMSF). GST was purified by affinity chromatography on a GSHepoxy activated Sepharose 6B affinity column (15 cm), prepared according to Mannervik and Guthenberg (1981). The column was preequilibrated with buffer A (22 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA) containing 300 mM NaCl. NaCl was also added to the cytosolic fraction before it was applied to the affinity column, at a flow rate of 4 ml/h, to a final concentration of 300 mM. After the cytosolic fraction was loaded, the column was run with buffer A containing 300 mM NaCl until the A 280 of the effluent fell under 0.01. GST activity was eluted with buffer B (buffer A containing 10 mM GSH, 10 mM 2-ME, and 25 AM PMSF). The portion of the
The specific activities of the separated isoenzymes towards the four substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), trans-4phenyl-3-buten-2-one (TPBO), and ethacrynic acid (EA) were determined as described by Habig et al. (1974). The GSH peroxidase activity of each isoenzyme was determined towards cumene hydroperoxide (CH). The assays consisted of 25 mM potassium phosphate buffer at pH 7.0, 0.2 mM NADPH, 1 mM GSH, 2 U of yeast GSH reductase, and 2 mM CH, and were read in a spectrophotometer for 15 min at 340 nm. GSH reductase activity was also assayed at 340 nm. The assay consisted of 0.1 M potassium phosphate buffer at pH 7.0, 1 mM EDTA, 2 mM oxidized GSH, and 0.2 mM NADPH. All assays were performed at room temperature and were run in triplicate. Each experiment was repeated at least three times. The substrates were added last after a 4-min preincubation period of the isoenzyme with GSH. Each assay contained about 0.1 Ag of purified enzyme protein. 2.5. Inhibitor sensitivities The sensitivity of the isoenzymes to certain inhibitors was studied according to Brophy et al. (1989) with minor alterations [i.e., Cibacron blue was dissolved in water, hematin in 0.1 M NaOH, and the other three inhibitors (chlorotriphenyltin, bromosulfophthalein, and lithocholic acid) were dissolved in absolute ethanol]. Inhibition is presented as I 50 values estimated by a computer program. GSH and CDNB were at 1 mM final concentration in the assay mixtures. 2.6. Gel filtration chromatography and PAGE About 1 ml of cytosolic fraction was applied onto a Sephadex G-75 column as described previously (Kostaropoulos and Papadopoulos, 1998). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was per-
Table 1 Purification protocol of cytosolic GST from whole body homogenate of adult worker bee A. mellifera macedonica Homogenate Bound fraction
Prt (mg/ml)
Activity
Specific activity
Purification
Yield
1.55F0.2 0.13F0.01
0.91F0.009 4.05F0.03
0.6F0.04 31.15F0.1
1 51.9
100 54.5
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Table 3 Substrate specificity of GST isoenzymes I and II purified from adult worker bee 1-Chloro-2,4-dinitrobenzene 2-Chloro-1,4-dinitrobenzene Ethacrynic acid Bromosulphophthalein trans-Phenyl-buten-one GSH reductase Cumene hydroperoxide
Isoenzyme I
Isoenzyme II
14.0F1.2 – 2.1F0.4 1.2F0.3 10.0F13.4 12.2F2.4 14.8F2.5
10.1F3.2 – – 1.05F0.2 31.5F6.8 27.4F6.7 1.2F0.6
The values represent micromoles per milligram per minute. Fig. 1. Chromatofocusing at pH range from 7 to 4 of affinity-purified GST from whole body homogenate of adult worker bee A. mellifera macedonica. Two isozymes were recovered: an alkaline isozyme with a pI of 7.5 and an acidic isozyme with a pI of 4.58.
formed on 10% resolving gel according to Laemmli (1970). The gels were stained with Coomassie Brilliant Blue. 2.7. Protein determination The protein was determined according to the method of Bradford (1976). Bovine serum albumin served as a standard. 2.8. Insect treatments 2.8.1. Low temperature Groups of 10–20 adult worker bees were kept in wellventilated boxes that were kept for 12 h in a refrigerated room, with a constant temperature of 6 8C. 2.8.2. Insecticide treatment Sublethal doses of decamethrin (20 and 40 ng/bee) and methyl paraoxon (1 ng/bee), as judged by LD50 experiments, were placed on the back of adult worker bees with a Hamilton microsyringe.
3. Results GST from A. mellifera macedonica was purified by affinity chromatography on a GSH-Sepharose column. The purification protocol is given in Table 1. Fifty to 55% of the total enzyme activity loaded on the column was recovered.
Table 2 Kinetic properties of GST isoenzymes I and II purified from adult worker bee K m GSH V max GSH K m CDNB V max CDNB
Isoenzyme I
Isoenzyme II
1.2F0.15 58.9F2.63 0.26F0.09 42.2F0.9
0.27F0.04 19.12F1.7 0.89F0.31 29.3F4.8
The values are given in micromolars and micromoles per milligram per minute, respectively.
The fractions containing the highest activity of pure enzyme, as judged by SDS-PAGE, were collected and concentrated by means of Amicon ultrafiltration cell. The purified enzyme was subsequently chromatofocused at a pH range between 4 and 7, in order to isolate the isoenzymes. As shown in Fig. 1, two main isoenzymes were found to be present: isoenzyme I with a corresponding pI value of 7.40 and isoenzyme II with a corresponding pI value of 4.58. For the study of the kinetic properties of the isoenzymes, we used the fractions with the highest activity. According to the results shown in Table 2, the alkaline isoenzyme (I) exhibits a significantly higher K m value towards GSH, higher V max GSH, and higher avidity towards CDNB than isoenzyme II. The activities of the two purified isoenzymes towards certain substrates are given in Table 3. Isoenzyme II showed no activity towards EA. It had a 70% reduced activity towards trans-phenyl-buten-one (TPBO) and 90% reduced activity towards CH than isoenzyme I. Bromosulphophthalein (BSP) served as substrate for both isoenzymes. Table 4 shows sensitivity towards selected inhibitors, expressed as I 50 values. Isoenzyme I appears to be more sensitive to all inhibitors studied, especially in the case of chlorotriphenyltin. Variations in the activity of the isoenzymes were observed under varying environmental conditions. As shown in Table 5, when the bees were exposed to low temperatures (6 8C) for 12 h, or were starved for 24 h, the activity corresponding to the peak of isoenzyme II increased, while that of isoenzyme I declined. Similar variations were observed when the insects were exposed to the insecticide methyl parathion (1 ng/bee) and decamethrin at 20 and 40 ng/bee. Worth noticing is the observation that
Table 4 Inhibitor sensitivity of purified isoenzymes from adult worker bee Cibacron blue Hematin Chlorotriphenyltin Bromosulphophthalein
Isoenzyme I
Isoenzyme II
0.053F0.01 0.13F0.013 0.33F0.04 0.053F0.03
0.073F0.02 1.81F0.55 6.4F0.8 1.24F0.37
The values given refer to the amount of inhibitor in millimoles required to cause 50% inhibition of the activity of the enzyme towards the model substrate CDNB.
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Table 5 Effects of various treatments on percentage of activity of the two isoenzymes of GST in adult worker bees Condition
Isoenzyme I
Isoenzyme II
Normal Freezing, 12 h Freezing, 24 h Starvation, 24 h Methyl parathion, 1 ng/bee Decamethrin, 20 ng/bee Decamethrin, 40 ng/bee
80.2F2.7 89.9F3.4 77.5F2.1 84.5F3.2 86.7F2.4 58.9F2.1 62.9F3.1
19.8F1.2 10.1F2.1 22.5F2.6 15.4F1.8 12.3F1.9 41.1F2.1 37.1F1.1
keeping the insects at low temperature for 24 h resulted in expression of the isoenzymes at rates similar to the controls. The purity of the GST derived from the body homogenate of A. mellifera (after it was passed through an affinity column) was tested by SDS-PAGE electrophoresis. By a combination of SDS-PAGE and gel filtration chromatography on Sephadex G-75, we estimated the molecular mass of the enzyme and its subunit composition. According to the results, the molecule of the enzyme is heterodimeric, consisting of two subunits with molecular masses of 29 and 25 kDa, respectively (Figs. 2 and 3).
4. Discussion According to a previous report (Papadopoulos et al., 2004), the number of GST isoenzymes present in the body of A. mellifera macedonica varies between two and four, depending on the developmental stage. The level of expression of the two main isoenzymes changes as the insect develops. While the acidic isoenzyme is the major one in the early developmental stages, alkaline isoenzymes are expressed more in the adult worker bee. This shift in expression towards the alkaline isoenzyme is accompanied by significant changes in the kinetic properties of total GST. In our present work, we purified GST from the adult worker bee by affinity chromatography and subsequently isolated the isoenzymes by chromatofocusing. The use of
Fig. 3. Determination of molecular mass by gel filtration on Sephadex G75 for cytosolic GST purified from whole body homogenate of adult worker bees (A. mellifera macedonica).
chromatofocusing for the separation of the isoenzymic forms of GST has been successfully used in previous investigations in mammals and insects. In the present work, an analytical column rather than a preparative column (with dimensions of 0.525 cm) was used, resulting in exceptional resolution and high repeatability. The two isoenzymes isolated from affinity-purified enzyme exhibited different kinetic properties. The alkaline isoenzyme exhibited higher K m towards GSH than the acidic isoenzyme; while towards the model substrate CDNB, it exhibited higher activity. Also, the V max of the alkaline isoenzyme for both substrates (GSH and CDNB) was higher than that estimated for the acidic isoenzyme. Differences also existed in the isoenzymes’ specificity towards various selected substrates, as well as the sensitivity towards selected inhibitors. The study of sensitivity towards certain inhibitors and the expressed activity towards certain substrates have been used in the past in order to classify the mammalian GSTs in four families (Mannervik et al., 1985; Meyer et al., 1992). More specifically, TBPO and DCNB are classified as A class marker substrates, while CH and EA are markers for a and k classes, respectively (Mannervik
Fig. 2. SDS-PAGE of cytoplasmic GST purified from whole body homogenates from adult worker bees.
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and Danielson, 1988). Regarding inhibitors, high susceptibility to Cibacron blue and chlorotriphenyltin suggests an isoenzyme belonging to the A class (Brophy et al., 1989). With these criteria, honey bee isoenzyme II does not seem to belong to any of the known mammalian classes, since it shows no reaction towards EA or DCNB. Isoenzyme I appears to be closer to either a or k class because it exhibits activity towards EA. However, most insect GSTs are usually classified as class theta (Wilce et al., 1995). According to our results, the alkaline isoenzyme has a faster turnover rate than the acidic isoenzyme. This characteristic apparently offers better protection to the worker bee against the various xenobiotics with which it may come in contact as it flies to flowers. The properties of the alkaline isoenzyme isolated in the present work offer a good explanation for the variation in the kinetic characteristics of the total enzyme observed during bee development, as detailed in our previous report (Papadopoulos et al., 2004). It is interesting to note that, based on our findings, exposure of the insect to low temperatures or starvation for 12 h leads to overexpression of the alkaline isoenzyme. The same increase was also observed with ethyl parathion. According to many investigators, the alkaline isoenzyme is involved in the protection against insecticides and therefore we would expect this isoenzyme to be expressed at higher rates during insecticide treatments. However, in our investigation, decamethrin injection caused increased expression of the acidic isoenzyme. A similar response was also observed in the larvae of the beetle Tenebrio molitor (Papadopoulos et al., 1999). Based on the results of this report, two main isoenzymes appear to be present in the adult worker bee, offering protection against various environmental parameters to which the insect may be exposed. These two isoenzymes are controlled independently, since their expression varies according to the environmental challenge. In spite of the lower number of isoenzymes, the alteration of the isoenzymic pattern caused by injection of a sublethal dose of insecticides is similar to that observed for other insects.
References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248 – 254. Brophy, P.M., Papadopoulos, A.I., Touraki, M., Coles, B., Korting, W., Barrett, J., 1989. Purification of cytosolic glutathione transferases from Schistocephalus solidus (pleroceroid): interaction with anthelmintics and products of lipid peroxidation. Mol. Biotechnol. Parasitol. 36, 187 – 196. Clark, A.G., 1989. The comparative enzymology of the glutathione Stransferases from non-vertebrate organisms. Comp. Biochem. Physiol., B 92, 419 – 446.
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Clark, A.G., Dick, G.L., Martindale, S.M., Smith, J.N., 1985. Glutathione S-transferases from the New Zealand grass grub, Costelytra zealandica: their isolation and characterization and the effect on their activity of endogenous factors. Insect Biochem. 15, 35 – 44. Fournier, D., Bride, J.M., Poirie, M., Berge, J.-B., Plapp Jr., F., 1992. Insect glutathione S-transferases: biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides. J. Biol. Chem. 267, 1840 – 1845. Gregus, Z., Varga, F., Schmelas, A., 1985. Age-development and inducibility of hepatic glutathione S-transferase activities in mice, rats, rabbits and guinea-pigs. Comp. Biochem. Physiol., C 80, 85 – 90. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130 – 7139. Hayaoka, T., Gauterman, W.C., 1982. Induction of glutathione S-transferase by phenobarbital and pesticides in various housefly strains and its effect on toxicity. Pestic. Biochem. Physiol. 17, 113 – 119. Hazelton, G.A., Lang, C.A., 1983. Glutathione S-transferase activities in the yellow-fever mosquito [Aedes aegypti (Louisville)] during growth and ageing. Biochem. J. 210, 281 – 287. Kostaropoulos, I., Papadopoulos, A.I., 1998. Glutathione S-transferase isoenzymes expressed in the three developmental stages of the insect Tenebrio molitor. Insect Biochem. Mol. Biol. 28, 901 – 909. Kostaropoulos, I., Mantzari, A.E., Papadopoulos, A.I., 1996. Alterations of some glutathione S-transferase characteristics during the development of Tenebrio molitor (Insecta: Coleoptera). Insect Biochem. Mol. Biol. 26, 963 – 969. Kostaropoulos, I., Papadopoulos, A.I., Metaxakis, A., Boukouvala, E., Papadopoulou-Mourkidou, E., 2001. Glutathione S-transferase in the defense against pyrethroids in insect. Insect Biochem. Mol. Biol. 31, 313 – 319. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of head bacteriophage T4. Nature 227, 680 – 685. Mannervik, B., Guthenberg, C., 1981. Glutathione transferase, (human placenta). Methods Enzymol. 77, 231 – 235. Mannervik, B., Danielson, U.H., 1988. Glutathione transferases—structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283 – 337. Mannervik, B., 2lin, P., Guthenberg, C., Jensson, H., Tahir, M.K., Warholm, M., Jornvall, H., 1985. Identification of three classes of cytosolic glutathione transferases common to several mammalian species: correlation between structural data and enzymatic properties. Proc. Natl. Acad. Sci. U. S. A. 82, 7202 – 7206. Meyer, D.J., Coles, B., Pemble, S.E., Fraser, G.M., Ketterer, B., 1992. Theta, a new class of glutathione transferases inferred from class-theta glutathione transferases cDNA sequences. Biochem. J. 287, 957 – 965. Motoyama, N., Dauterman, W.C., 1980. Glutathione S-transferases: their role in the metabolism of organophosphorus insecticides. Rev. Biochem. Toxicol. 2, 49 – 69. Papadopoulos, A.I., Stamkou, E.I., Kostaropoulos, I., PapadopoulouMourkidou, E., 1999. Effect of organophosphate and pyrethroid insecticides on the expression of GSTs from Tenebrio molitor larvae. Pestic. Biochem. Physiol. 63, 26 – 33. Papadopoulos, A.I., Boukouvala, E., Kakaliouras, G., Kostaropoulos, I., Papadopoulou-Mourkidou, E., 2000. Effect of organophosphate and pyrethroid insecticides on the expression of GSTs from Tenebrio molitor pupae. Pestic. Biochem. Physiol. 68, 26 – 33. Papadopoulos, A.I., Polemitouc, E., Laifi, P., Yiangou, A., Tananaki, T., 2004. Glutathione S-transferase in the developmental stages of the insect Apis mellifera macedonica. Comp. Biochem. Physiol., C 139, 87 – 92. Reidy, G.F., Rose, H.A., Visetson, S., Murray, M., 1990. Increased glutathione S-transferase activity and glutathione content in an insecticide-resistant strain of Tribolium castaneum (Herbst). Pestic. Biochem. Physiol. 36, 269 – 276. Wilce, M.C.J., Board, P.G., Feil, S.C., Parker, M.W., 1995. Crystal structure of a theta-class glutathione transferase. EMBO J. 14, 2133 – 2143.
Glutathione S-transferase in the insect Apis mellifera macedonica Kinetic characteristics and effect of stress on the expression of GST isoenzymes in the adult worker bee Athanasios I. Papadopoulosa,*, Irene Polemitoua, Pshychoula Laifia, Astero Yiangoua, Chrysoula Tananakib a
Laboratory of Animal Physiology, Department of Zoology, Faculty of Science, School of Biology, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece b Laboratory of Apiculture–Sericulture, Department of Agriculture, Geotechnical Sciences, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece
Received 2 June 2004; received in revised form 21 September 2004; accepted 22 September 2004
Abstract The glutathione S-transferase present in the adult worker bee Apis mellifera macedonica was purified and analyzed for its physicochemical and kinetic properties. The enzyme is heterodimeric with subunit molecular masses of 29 and 25 kDa, respectively. Two main isoenzymes with distinct kinetic properties are present, with isoelectric points of 7.40 for the alkaline and 4.58 for the acidic forms, respectively. The two enzymes are induced independently by factors such as insecticide treatments and environmental conditions, including low temperatures or starvation. D 2004 Elsevier Inc. All rights reserved. Keywords: Glutathione S-transferase; Adult worker bee; Apis mellifera macedonica; Detoxification; Isoenzymes; Induction
1. Introduction Glutathione S-transferases (GSTs) are present in almost all animals and in most of them in multiple isoenzymic forms, constituting a significant intracellular mechanism of detoxification. The enzymes catalyse the conjugation of a large variety of compounds bearing an electrophilic site, with reduced glutathione (GSH) (Mannervik and Danielson, 1988). In insects, they represent a very interesting detoxification mechanism due to their involvement in tolerance to insecticides (Motoyama and Dauterman, 1980; Clark et al., 1985; Fournier et al., 1992; Kostaropoulos et al., 2001). Several authors have noted that increases in both GSH content and GST activity are
* Corresponding author. Tel.: +30 2310 998359; fax: +30 2310 998269. E-mail address: [email protected] (A.I. Papadopoulos). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.09.010
associated with resistance to organophosphorus insecticides (Reidy et al., 1990; Clark, 1989). The multiple isoenzymic forms of GST are distinguished by differences in structure and catalytic properties. The expression of isoenzymes depends on many internal and external factors. Age-dependent alteration of GST activities has been demonstrated in both vertebrates and invertebrates (Gregus et al., 1985; Hazelton and Lang, 1983; Kostaropoulos et al., 1996). Alteration of GST expression induced by various substances in insets has also been reported (e.g., food quality and administration of certain insecticides) (Hayaoka and Gauterman, 1982; Papadopoulos et al., 1999; Papadopoulos et al., 2000; Kostaropoulos et al., 2001). Significant alterations in the isoenzymic profile, leading to the suggestion that the multiple GST isoenzymes present in insects are regulated independently, have also been reported as a result of stress, caused by a variety of factors (Papadopoulos et al., 1999, 2001).
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In our previous work (Papadopoulos et al., 2004), we detailed alterations in isoenzymic pattern during the development of the honeybee, Apis mellifera macedonica. In the present paper, we report on the purification of GST from the adult worker bee, its kinetic characteristics, its molecular mass, the isolation of isoenzymes from purified enzyme, and their kinetic characteristics. We also report on the effect of environmental parameters upon the expression of the isoenzymic profile.
activity that remained bound to the column was eluted with high salt (3 M NaCl in buffer A, designated as buffer C). The purification procedure was carried out at 4 8C. The protein fractions eluted by buffers B and C were pooled and concentrated twice against 10 mM potassium phosphate buffer at pH 6.5 in an Amicon ultrafiltration cell, and the concentrate was either subjected to isoelectric focusing (IEF) or chromatofocusing at a pH range 7–4, as recommended (Pharmacia) on a 0.36-cm column. 2.4. Substrate specificity
2. Materials and methods 2.1. Chemicals and reagents The reagents for chromatofocusing, epoxy-activated Sepharose 6B, and the low-molecular-weight and broad pI calibration markers were purchased from Pharmacia (Uppsala, Sweden). All other chemicals were from Sigma (St. Louis, MO, USA). 2.2. Insects Honeybees (A. mellifera macedonica) were supplied from the apiary of the laboratory Apiculture in Aristotle University of Thessaloniki. 2.3. Preparation of cytosolic fractions and purification of the enzyme The cytosolic fraction was prepared from whole bodies of 10–15 individuals, as described previously (Kostaropoulos et al., 1996), using a modified homogenization medium (10 mM potassium phosphate buffer, pH 7.4, 0.25 M sucrose, 0.2 mM EDTA, 10 mM 2-mercaptoethanol (ME), and 25 AM PMSF). GST was purified by affinity chromatography on a GSHepoxy activated Sepharose 6B affinity column (15 cm), prepared according to Mannervik and Guthenberg (1981). The column was preequilibrated with buffer A (22 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA) containing 300 mM NaCl. NaCl was also added to the cytosolic fraction before it was applied to the affinity column, at a flow rate of 4 ml/h, to a final concentration of 300 mM. After the cytosolic fraction was loaded, the column was run with buffer A containing 300 mM NaCl until the A 280 of the effluent fell under 0.01. GST activity was eluted with buffer B (buffer A containing 10 mM GSH, 10 mM 2-ME, and 25 AM PMSF). The portion of the
The specific activities of the separated isoenzymes towards the four substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), trans-4phenyl-3-buten-2-one (TPBO), and ethacrynic acid (EA) were determined as described by Habig et al. (1974). The GSH peroxidase activity of each isoenzyme was determined towards cumene hydroperoxide (CH). The assays consisted of 25 mM potassium phosphate buffer at pH 7.0, 0.2 mM NADPH, 1 mM GSH, 2 U of yeast GSH reductase, and 2 mM CH, and were read in a spectrophotometer for 15 min at 340 nm. GSH reductase activity was also assayed at 340 nm. The assay consisted of 0.1 M potassium phosphate buffer at pH 7.0, 1 mM EDTA, 2 mM oxidized GSH, and 0.2 mM NADPH. All assays were performed at room temperature and were run in triplicate. Each experiment was repeated at least three times. The substrates were added last after a 4-min preincubation period of the isoenzyme with GSH. Each assay contained about 0.1 Ag of purified enzyme protein. 2.5. Inhibitor sensitivities The sensitivity of the isoenzymes to certain inhibitors was studied according to Brophy et al. (1989) with minor alterations [i.e., Cibacron blue was dissolved in water, hematin in 0.1 M NaOH, and the other three inhibitors (chlorotriphenyltin, bromosulfophthalein, and lithocholic acid) were dissolved in absolute ethanol]. Inhibition is presented as I 50 values estimated by a computer program. GSH and CDNB were at 1 mM final concentration in the assay mixtures. 2.6. Gel filtration chromatography and PAGE About 1 ml of cytosolic fraction was applied onto a Sephadex G-75 column as described previously (Kostaropoulos and Papadopoulos, 1998). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was per-
Table 1 Purification protocol of cytosolic GST from whole body homogenate of adult worker bee A. mellifera macedonica Homogenate Bound fraction
Prt (mg/ml)
Activity
Specific activity
Purification
Yield
1.55F0.2 0.13F0.01
0.91F0.009 4.05F0.03
0.6F0.04 31.15F0.1
1 51.9
100 54.5
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Table 3 Substrate specificity of GST isoenzymes I and II purified from adult worker bee 1-Chloro-2,4-dinitrobenzene 2-Chloro-1,4-dinitrobenzene Ethacrynic acid Bromosulphophthalein trans-Phenyl-buten-one GSH reductase Cumene hydroperoxide
Isoenzyme I
Isoenzyme II
14.0F1.2 – 2.1F0.4 1.2F0.3 10.0F13.4 12.2F2.4 14.8F2.5
10.1F3.2 – – 1.05F0.2 31.5F6.8 27.4F6.7 1.2F0.6
The values represent micromoles per milligram per minute. Fig. 1. Chromatofocusing at pH range from 7 to 4 of affinity-purified GST from whole body homogenate of adult worker bee A. mellifera macedonica. Two isozymes were recovered: an alkaline isozyme with a pI of 7.5 and an acidic isozyme with a pI of 4.58.
formed on 10% resolving gel according to Laemmli (1970). The gels were stained with Coomassie Brilliant Blue. 2.7. Protein determination The protein was determined according to the method of Bradford (1976). Bovine serum albumin served as a standard. 2.8. Insect treatments 2.8.1. Low temperature Groups of 10–20 adult worker bees were kept in wellventilated boxes that were kept for 12 h in a refrigerated room, with a constant temperature of 6 8C. 2.8.2. Insecticide treatment Sublethal doses of decamethrin (20 and 40 ng/bee) and methyl paraoxon (1 ng/bee), as judged by LD50 experiments, were placed on the back of adult worker bees with a Hamilton microsyringe.
3. Results GST from A. mellifera macedonica was purified by affinity chromatography on a GSH-Sepharose column. The purification protocol is given in Table 1. Fifty to 55% of the total enzyme activity loaded on the column was recovered.
Table 2 Kinetic properties of GST isoenzymes I and II purified from adult worker bee K m GSH V max GSH K m CDNB V max CDNB
Isoenzyme I
Isoenzyme II
1.2F0.15 58.9F2.63 0.26F0.09 42.2F0.9
0.27F0.04 19.12F1.7 0.89F0.31 29.3F4.8
The values are given in micromolars and micromoles per milligram per minute, respectively.
The fractions containing the highest activity of pure enzyme, as judged by SDS-PAGE, were collected and concentrated by means of Amicon ultrafiltration cell. The purified enzyme was subsequently chromatofocused at a pH range between 4 and 7, in order to isolate the isoenzymes. As shown in Fig. 1, two main isoenzymes were found to be present: isoenzyme I with a corresponding pI value of 7.40 and isoenzyme II with a corresponding pI value of 4.58. For the study of the kinetic properties of the isoenzymes, we used the fractions with the highest activity. According to the results shown in Table 2, the alkaline isoenzyme (I) exhibits a significantly higher K m value towards GSH, higher V max GSH, and higher avidity towards CDNB than isoenzyme II. The activities of the two purified isoenzymes towards certain substrates are given in Table 3. Isoenzyme II showed no activity towards EA. It had a 70% reduced activity towards trans-phenyl-buten-one (TPBO) and 90% reduced activity towards CH than isoenzyme I. Bromosulphophthalein (BSP) served as substrate for both isoenzymes. Table 4 shows sensitivity towards selected inhibitors, expressed as I 50 values. Isoenzyme I appears to be more sensitive to all inhibitors studied, especially in the case of chlorotriphenyltin. Variations in the activity of the isoenzymes were observed under varying environmental conditions. As shown in Table 5, when the bees were exposed to low temperatures (6 8C) for 12 h, or were starved for 24 h, the activity corresponding to the peak of isoenzyme II increased, while that of isoenzyme I declined. Similar variations were observed when the insects were exposed to the insecticide methyl parathion (1 ng/bee) and decamethrin at 20 and 40 ng/bee. Worth noticing is the observation that
Table 4 Inhibitor sensitivity of purified isoenzymes from adult worker bee Cibacron blue Hematin Chlorotriphenyltin Bromosulphophthalein
Isoenzyme I
Isoenzyme II
0.053F0.01 0.13F0.013 0.33F0.04 0.053F0.03
0.073F0.02 1.81F0.55 6.4F0.8 1.24F0.37
The values given refer to the amount of inhibitor in millimoles required to cause 50% inhibition of the activity of the enzyme towards the model substrate CDNB.
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Table 5 Effects of various treatments on percentage of activity of the two isoenzymes of GST in adult worker bees Condition
Isoenzyme I
Isoenzyme II
Normal Freezing, 12 h Freezing, 24 h Starvation, 24 h Methyl parathion, 1 ng/bee Decamethrin, 20 ng/bee Decamethrin, 40 ng/bee
80.2F2.7 89.9F3.4 77.5F2.1 84.5F3.2 86.7F2.4 58.9F2.1 62.9F3.1
19.8F1.2 10.1F2.1 22.5F2.6 15.4F1.8 12.3F1.9 41.1F2.1 37.1F1.1
keeping the insects at low temperature for 24 h resulted in expression of the isoenzymes at rates similar to the controls. The purity of the GST derived from the body homogenate of A. mellifera (after it was passed through an affinity column) was tested by SDS-PAGE electrophoresis. By a combination of SDS-PAGE and gel filtration chromatography on Sephadex G-75, we estimated the molecular mass of the enzyme and its subunit composition. According to the results, the molecule of the enzyme is heterodimeric, consisting of two subunits with molecular masses of 29 and 25 kDa, respectively (Figs. 2 and 3).
4. Discussion According to a previous report (Papadopoulos et al., 2004), the number of GST isoenzymes present in the body of A. mellifera macedonica varies between two and four, depending on the developmental stage. The level of expression of the two main isoenzymes changes as the insect develops. While the acidic isoenzyme is the major one in the early developmental stages, alkaline isoenzymes are expressed more in the adult worker bee. This shift in expression towards the alkaline isoenzyme is accompanied by significant changes in the kinetic properties of total GST. In our present work, we purified GST from the adult worker bee by affinity chromatography and subsequently isolated the isoenzymes by chromatofocusing. The use of
Fig. 3. Determination of molecular mass by gel filtration on Sephadex G75 for cytosolic GST purified from whole body homogenate of adult worker bees (A. mellifera macedonica).
chromatofocusing for the separation of the isoenzymic forms of GST has been successfully used in previous investigations in mammals and insects. In the present work, an analytical column rather than a preparative column (with dimensions of 0.525 cm) was used, resulting in exceptional resolution and high repeatability. The two isoenzymes isolated from affinity-purified enzyme exhibited different kinetic properties. The alkaline isoenzyme exhibited higher K m towards GSH than the acidic isoenzyme; while towards the model substrate CDNB, it exhibited higher activity. Also, the V max of the alkaline isoenzyme for both substrates (GSH and CDNB) was higher than that estimated for the acidic isoenzyme. Differences also existed in the isoenzymes’ specificity towards various selected substrates, as well as the sensitivity towards selected inhibitors. The study of sensitivity towards certain inhibitors and the expressed activity towards certain substrates have been used in the past in order to classify the mammalian GSTs in four families (Mannervik et al., 1985; Meyer et al., 1992). More specifically, TBPO and DCNB are classified as A class marker substrates, while CH and EA are markers for a and k classes, respectively (Mannervik
Fig. 2. SDS-PAGE of cytoplasmic GST purified from whole body homogenates from adult worker bees.
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and Danielson, 1988). Regarding inhibitors, high susceptibility to Cibacron blue and chlorotriphenyltin suggests an isoenzyme belonging to the A class (Brophy et al., 1989). With these criteria, honey bee isoenzyme II does not seem to belong to any of the known mammalian classes, since it shows no reaction towards EA or DCNB. Isoenzyme I appears to be closer to either a or k class because it exhibits activity towards EA. However, most insect GSTs are usually classified as class theta (Wilce et al., 1995). According to our results, the alkaline isoenzyme has a faster turnover rate than the acidic isoenzyme. This characteristic apparently offers better protection to the worker bee against the various xenobiotics with which it may come in contact as it flies to flowers. The properties of the alkaline isoenzyme isolated in the present work offer a good explanation for the variation in the kinetic characteristics of the total enzyme observed during bee development, as detailed in our previous report (Papadopoulos et al., 2004). It is interesting to note that, based on our findings, exposure of the insect to low temperatures or starvation for 12 h leads to overexpression of the alkaline isoenzyme. The same increase was also observed with ethyl parathion. According to many investigators, the alkaline isoenzyme is involved in the protection against insecticides and therefore we would expect this isoenzyme to be expressed at higher rates during insecticide treatments. However, in our investigation, decamethrin injection caused increased expression of the acidic isoenzyme. A similar response was also observed in the larvae of the beetle Tenebrio molitor (Papadopoulos et al., 1999). Based on the results of this report, two main isoenzymes appear to be present in the adult worker bee, offering protection against various environmental parameters to which the insect may be exposed. These two isoenzymes are controlled independently, since their expression varies according to the environmental challenge. In spite of the lower number of isoenzymes, the alteration of the isoenzymic pattern caused by injection of a sublethal dose of insecticides is similar to that observed for other insects.
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