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Camel brain glutathione S-transferase purification, properties, regional and subcellular distribution
Comp. Biochem. PhysioL Vol. 93B, No. 2, pp. 333-338, 1989 Printed in Great Britain
0305-0491/89 $3.00+ 0.00 © 1989PergamonPress pie
CAMEL BRAIN GLUTATHIONE S-TRANSFERASE PURIFICATION, PROPERTIES, REGIONAL AND SUBCELLULAR DISTRIBUTION A. A. H U N A m * and Q. ASA'D Laboratory of Biochemistry and Molecular Biology, Department of BiologicalSciences, Yarmouk University,Irbid,Jordan. (Tel:0271100) (Recewed22 June 1988)
Abstract--l. Camel brain glutathione S-transferase was purified by glutathione-linked agarose affinity column and the different isozymes were separated by chromatofocusing. 2. The basic isozymes which comprise 45% of the total activity were immunologicallyindistinguishable from the near-neutral isozymes which constitute 55% of the activity. 3. Some differenceswere detectable among the basic and near-neutral isozymes in relation to substrate specificities and subunit composition. 4. Biochemical and immunological quantification of glutathione S-transferase revealed the presence of the enzyme in all camel brain regions tested and subcellular fractions. 5. The pons had the highest concentration of the enzyme and the cortex had the lowest, while more than 88% of the enzyme was present in the cytosoL
INTRODUCTION
MATERIALS
The giutathione S-transferases (EC 2.5.1.18) are a family of multifunctional proteins which catalyse the conjugation of glutathione with a broad variety of electrophilic and lipophilic compounds, many of which are toxic, mutagenic and carcinogenic (Chasseud, 1979). In mammals the transferases are all dimeric proteins of mol. wt approx. 50,000 and composed of multiple forms which are distinguished from one another by their subunit compositions and electrophoretic mobilities (Jakoby and HHabig, 1980). In guinea pig brain four major forms of the enzyme have been purified (Di Llio et aL, 1982) while three forms have been identified in human brain (Theodore et aL, 1985). Moreover, Polidoro et aL (1984) observed a remarkable difference in the nature and number of various forms of glutathione S-transferase obtained from brain cortex of cattle, sheep, mouse, guinea pig and human. In our previous work (I-Iunaiti and Owais, 1985; Hunaiti and Abu Khalaf, 1986) we demonstrated the presence of glutathione S-transferase in camel brain homogenate. However, little information is available concerning the subcellular and regional distribution of the enzyme in the brains of various animals including camel. An attempt to relate the presence of glutathione S-transferase in the brain to its role in the detoxification of various neurotoxins, implies a knowledge of the level of this enzyme in different subcellular and regional fractions of the brain. Therefore, in this paper we report on the purification of glutathione S-transferase from camel brain homogenate and the biochemical and immunological quantification of this enzyme in various subcellular and regional fractions of camel brain. *Author to whom correspondence should be addressed.
ANDMETHODS
Materials All biochemicalsused in the present study were purchased from Siama Chemical Company (USA) except for polybuffer exchangers which were from Pharmacia (Sweden)and l-chioro-2,4-dinitrobenzene and trans-4-phenyl-3-buten-2one were from Aldrich (England). Brain of l-year-old onehumped camel (Camelua dromedar/us) was obtained from a local slaughter house immediately after killing the animal and transported under ice to the laboratory. Enzyme assay The glutathione S-trausferase activity toward pnitrobenzylchioride, 1,2-epoxy-3-(p-nitrophenoxy)propane, l-chloro-2,4-dinitrobenzene, ethacrynic acid and trans4-phenyl-3-buten-2-one was determined spectrophotometrically using a Pye Unicam SP 3-400 Spectrophotometer essentially as described by Habig et ai. (1974). Enzyme activity was expressed as /zmol/min and specific activity as ~mol/min/mg protein. Protein concentrations were determined by the Bradford method (Bradford, 1976) using reagents from Bio-Rad (USA). Preparation of crude extract Fresh camel brain (316g) was washed with ice-cold distilled water and sliced into small pieces. The tissues were homogenized in 474ml of 0.1 M potassium phosphate buffer pH 7.0 containing 1.4 mM ~-mereaptoethanol using a Waring blender operating at maximum speed for 1 rain. All further steps were performed at 4°C. The homogenate was centrifuged at 37,000g for 30 min, then the supernatant was filtered through a plug of glass wool to remove floating particles. Fractionation of camel brain~ Cerebral cortex, cerebellum, cerebrum, medulla oblongata, thalrnus, hypothalmus and pons were carefully excised from the whole camel brain and crude extracts of the various regions were prepared essentially as described by Eichberg and Karnosky (1969). White matter (100g) of camel brain was used to prepare myelin fraction as described by Norton (1974). Transverse section (100 g) of
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fresh camel brain was homogenized as described above and centrifuged at 1000g for 10 vain. The pellet was suspended in (1:1.5 w/v) 0.1 M potassium phosphate buffer pH 7.2, recentrifuged, resuspended and labelled as nuclear fraction. The original supematants were further fractionated by centrifugation at 1200g for 15 rain and the resulting pellet was washed twice with the same buffer and labelled as mitocliondrial fraction. Microsomal and cytosolic fractions were obtained by further centrifugation of the supematant at 105,000g for 60rain.
Purification of glutathione S-transferase by affinity chromatography The filtrate obtained from the previous step was applied to a column (1 x 10 cm) packed with glutathione linked to epoxy activated agarose at a flow rate of 10 ml/hr after a pre-equilibration with 22 mM potassium phosphate buffer pH 7.0. The column was washed thoroughly with the same buffer containing 0.1 M NaCI until the protein absorbance at 280 nm of the effluent returned to zero. Subsequently the bound enzyme was eluted with 50mM Tris-HCl buffer, pH 9.2 containing 10 mM reduced glutathione. The fractions (2 ml each) having the highest enzymatic activity were pooled and dialysed overnight against distilled water (300 vol. two changes). The dialyzed sample was concentrated to 4 ml using an Amicon Ultra filtration cell equipped with PM 30 membrane. The concentrated enzyme was subsequently diluted with 15 volumes of double distilled water and concentrated to 4 ml.
Chromatofocusing The concentrated sample (28 mg) was applied to a chromatofocusing column (0.9 x 40 cm) packed with polybuffer exchanger 94 which had been equilibrated with 15 bed volumes of the starting buffer (0.025 M ethanolamine-HCl pH 9.4). The different isozymes were ¢luted with polybuffer 96 (diluted 1 : I0 with distilled water and adjusted to pH 6.0 with acetic acid), until the pH of eluent reached 6.0. Then a second eluent polybuffer 64 (diluted 1 : 8 and adjusted to pH 4 with HCI) was used. The flow rate was 18 ml/hr and 0.8 ml fractions were collected. Each enzymatic peak was collected and dialysed overnight against 0.1 M potassium phosphate buffer 7.2. Polybuffer was removed from all samples by affinity chromatography as described above.
Electrophoresis Analytical gel electrophoresis of the purified enzyme (20-30 #g) was carded out at 4°C in 15% SDSpolyacrylamide gel as described by Laemmli (1970).
Antibody production Antibodies against affinity purified glutathione Stransferases and isoenzyme C: (pI 7.9) and C6 (pI 7.2) were raised in three different rabbits. In each case about 400 #g protein was thoroughly mixed with Frennd's complete adjuvant (1 : 1 v/v) and the resulting emulsion (0.8 ml) was
injected intramuscularly into three different sites of one of the large hind thighs of the rabbit. Two weeks later a similar injection in Freund's incomplete adjuvant was made. After 3 weeks, the rabbits were bled and the antisera were collected.
Immunodiffusion Ouchteriony double diffusion analysis was performed on microscopic slides with 1.5% agarose in 75 mM barbital buffer pH 8.6 (Ouchterlony, 1958). After 2448 hr the nonagglutinated proteins were removed by copious washing with 0.15MNaC1 and the immunopr~pitin lines were visualized by staining with Coomassie Brilliant Blue R-250.
Rocket immunoelectrophoresis Rocket immunoelectrophoresis was performed on (7.5 x 5cm) preheated glass plates loading with 1.5% agarose which was prepared in 75raM barbital buffer pHS.5, 5mM EDTA, 0.02% sodium azide and 0.9% NaC1 essentially as described (Laurell and McKay, 1981). Electrophoresis was performed at 4°C and 10 V/cm using BRL horizontal electrophoresis. Rockets were usually formed after 4-6 hr. RESULTS
Purification of glutathione S-transferase from camel brain Crude extracts prepared from camel brain contained considerable amounts of glutathione Stransferase activity about 20 units/gram wet wt. The specific activity towards the most c o m m o n l y used substrate l,chloro-2,4-dinitrobenzene ranged from 1.8 to 2.3 units/rag protein. Application o f the 37,000g supematant onto a glutathione agaros¢ affinity column resulted in retaining all the enzymatic activity but not the bulk o f the protein. Elution of the column with 10 m M glutathione recovered more than 62% of the applied activity (Table l) in a single sharp symmetrical peak o f activity. It was estimated that glutathione Stransferase activity represents about 1.4% o f the total extractable protein of camel brain. When the glutathione S-transferase obtained from the affinity column was applied to the chromatofocusing column, the enzyme was resolved into at least eight distinct peaks ( F i g . 1). The five basic isoenzymes which were eluted between p H 8.0 and p H 7.4 accounted for about 45% of the total enzymatic activity towards 1-chloro-2,4-dinitrobenzene. The major basic isoenzyme with pI 7.9 constituted about 70% of the total basic isoenzymes. The three near-neutral isoenzymes which were eluted between p H 7 . 3
Table 1. Purification of glutathione S-transferase from camel brain Specific Total Activity activity Purification Yield Purification step protein (total units) (units/mg) (fold) (%) Crude extract 3316.8 6168 1.859 1.0 100 Affinity chromatography 28.0 3840 137.0 73.6 62 Chromatofocusing Peak C1 (pI 7.95) 0.04 1.1 27.5 14.8 0.0178 Peak C2 (pl 7.85) 1.05 158.0 150.0 80.6 2.56 Peak C3 (pI 7.8) 0.61 49.0 78.0 42.1 0.79 Peak C4 (pl 7.6) 0.133 19.0 140.0 75.3 0.308 Peak C5 (pI 7.4) 0.0275 6.0 54.5 29.3 0.097 Peak C6 (pI 7.3) 0.45 83 180.0 97.0 1.345 Peak C7 (pl 7.0) 0.182 40.6 166 89.7 0.65 Peak C8 (pI 6.9) 1.5 160.0 106.0 57.2 2.5 Enzyme was purified from 316 g (wet wt) of camel brain by the methods described in Materials and Methods.
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Fig. 1. Chromatofocusingprofile of purified giutathione S-transferase from camel brain. Experimental details are given in the text. and pH 6.9 exhibited about 55% of the enzymatic activity. No acidic isoenzymes were observed even when five different camel brains were analysed by this method.
Biochemical properties of camel brain glutathione S-transferase Figure 2 shows the SDS-polyacrylamide gel electrophoresis of the enzyme preparations obtained during the purification process. The affinity purified enzyme exhibited two protein bands at lit, 26,300 and 24,000 (Fig. 2, lane 7). The individual peaks of glutathione S-transferase obtained from chromatofocusing column were also analysed by SDSelectrophoresis. The near-neutral peaks (]6 and C7 revealed the presence of a single protein band at M, 26,300 (Fig. 2, lanes 1 and 2). The basic isozymes C3 and C, exhibited two protein bands at 26,300 and 24,000 (Fig. 2, lanes 3 and 4), while the basic Cs isozymes consisted primarily of the lower protein band M, 24,000 and faint band at M, 26,300 (Fig. 2, lane 6). Other isoenzymes were not analysed. The native mol. wt of the purified enzyme as determined by calibrated sephadex G-100 column was about 52,000. Thus the structure of camel brain glutathione S-transferase is a dimeric protein. Substrate specificities of the major camel brain glutathione S-transferase isozymes were determined and the results are shown in Table 2. The preferred substrate for the tested isozymes was 1-chloro-2,4-dinitrobenzene. Significant differences
were also observed between the basic and the near-neutral isozymes towards different substrates. The basic form had higher specific activity for 1,2-epoxy-3(p-nitropbenoxy)propane. The nearneutral isozymes but not the basic isozymes exhibited glutathione peroxide activity (Table 2).
Stability The freezing and thawing process of the purified enzyme caused a loss of 10-15% of the activity, while incubation of the enzyme at 60°C for 4 rain resulted in 90% loss of the activity. Storage of the purified enzyme for 1 yr at -20°C caused loss of about 20% of the initial activity with concurrent appearance of four protein bands on SDS-gel electrophoresis. Immunological properties of camel brain glutathione S-transferase Immunotitration showed that when antibodies raised against the major C2(pI 7.9), Cr(pI 7.2) and affinity purified pool glutathion¢ S-transferase were incubated with the respective enzyme, the enzymatic activity towards l-chloro-2,4-dinitrobenzene was inhibited in a concentration-dependent manner. Ouchterlony double diffusion analysis showed that antibodies raised against camel brain glutathione S-transferase cross-reacted only with camel brain homogenate, but not with homogenates prepared from rabbit, mouse, rat, guinea pig, sheep and cow brains (Fig. 3). Similarly the antibodies were also
Table 2. Substrate spccificities of different camel brain glutathione S-transferase isozymes Glutathione S transferase activity (~mol/min/mg) Major basic isozyme Substrate Affinity pool (pI 7.9) 1-Chloro-2,4-dinitrobenzene 65.6 58.14 Nitrobenzyl chloride 8.87 0.75 Ethacrynic acid 5.68 7.1 1,2-Epoxy-p-nitrophenoxy propane 2.3 1.14 Trans-4-phenyl-3-buten-2-one 0.0015 ND Cumene hydroperoxide* ND ND Experimental details are indicated in the text. ND, not detected. *Glutathione peroxidase activity.
Major near neutral isozyme (pI 6.9) 91.6 3.31 0.84 6.3 ND 0.05
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A.A. HUNAITIand Q. ASA'D
Fig. 2. SDS-polyacrylamide gel electrophoresis (15%) of camel brain glutathione S-transferases. Lane 1 C6 (pI 7.2) isozyme, lane 2 C7 (pI 6.9) isozyme, lane 3 (pI 7.3) isozyme, lane 4 CA (pI 7.6) isozyme, lane 5 C6 isozyme, lane 6 C5 (pI 7.4) isozyme, lane 7 affinity pool enzyme. Lane 8 protein standards {bovine serum albumin 66,000, ovalbumin 45,000, glyceraldehyde 3-phosphate dehydrogenase 36,000, carbonic anbydrase 29,000, trypsinogen 24,000, trypsin inhibitor 20, 100 and/~-lactoalbumin 14,400). In each case about 10/~g proteins were loaded. shown to react with crude extracts prepared from various camel organs such as lung, heart, retina, kidney and liver (data not shown).
Regional and subcellular distribution of the enzyme When the various regions of camel brain were carefully separated and the glutathione S-transferase activity was measured in each region, some
differences in the total and specific activity were observed (Table 3). The highest specific activity was found in the pons while the lowest specific activity was associated with the cortex. The total enzyme activity was highest in the cerebrum and lowest in the medulla oblongata. Moreover, the distribution of glutathione S-transferase in different regions of camel brain was quantified by an immunological technique.
Fig. 3. Ouchterlony double-diffusion analysis of glutathione S-transferases in brain cytosol of several mammalian species. Centre wells contain 0.25 mg of antibody raised against atfmity purified camel enzyme. Outer wells contain 0.5 mg of brain cytosols of C, camel', RAB, rabbit; M, mouse; RAT; GP, guinea pig; S, sheep, Cow.
Camel glutathione S-transferase
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Table 3. Biochemicaland immunologicalquantitation of giutathione S-transferasein different regions of camel brain* Total giutathione Total activity Specificactivity S-transferase Brain region 0zmol/min) ~mol/min/mg) 0~g/mg protein)$ Cerebellum 13,158 3.62 t.25 Cortex 513 1.43 0.75 Cerebrum 3120 3.42 1.38 Medulla oblongata 95 3.85 3.2 Thalmus 680 3.65 2.3 Hypothalmus 975 3.32 2.0 Pons 385 6.64 3.5 Remainderi" 3540 3.58 1.3 *Prepared from 635 g of camel brain tissue. ")'Remainderrefers to other parts of the brain not already specifiedin the table. :~Determinedby rocket immunoelectrophoresis. Table 4. Subcellulardistributionof glutathione S-transferas¢in camel brain* Specific Total .giutathione Activity activity S-transferase ~mol/min) (/~mol/min/mg) ~g/mg protein)t Fraction Nuclear fraction 29.5 0.588 0.4 Mitochondrial fraction 39.7 0.629 0.32 Mierosomalfraction 45.9 0.316 0.068 Cytosol 92.5 61.5 12.5 Myelin sheath 39.37 1.89 3.84 *Prepared from 300 g of camel brain tissue. tDetermined by rocket electrophoresis. dimeric protein with a native mol. wt of 52,000. The subunit composition and molecular weights are in close agreement with those reported for other species (Jakoby and Habig, 1980; Theodore et al., 1985; Mannervik, 1985). Isoelectric fractionation of camel brain giutathione S-transferase resulted in a distinguished isozyme pattern with basic forms comprising about 45% of the total activity and a near-neutral comprising about 55% of the activity and complete absence of acidic forms. This is quite different from the isozymes pattern of human brain in which about 85% of the activity was due to acidic forms (Theodore et al., 1985). It appears that the antigenic determinants present in camel brain glutathione S-transferase are distinct from those present in brain cytosols of mice, guinea pigs, rabbits, cows and sheep. Furthermore, the immunological studies do suggest a close relationship between the different forms of glutathione S-transferase in camel brain tissue and the various forms of the enzyme in camel lung, retina, heart and liver. DISCUSSION To find out whether the whole brain or a specific Our study clearly demonstrates that camel brain part of it engaged in the detoxification of endocontains considerable amounts of glutathione S- geneous and/or exogenous eleetrophilic toxicants, transferase. The content of this enzyme in camel biochemical and immunological quantification of brain is about 20 units/g wet wt. This level is similar the enzyme were employed. The results of such an to the previously reported value for human brain investigated clearly showed the distribution of the which was about 12 units/g wet wt (Theodore et al., enzyme in various brain regions. These findings 1985). The specific activity of the enzyme in crude are in close agreement with those reported for rat extracts from camel brain ranges from 1.8 to 2.3 brain enzyme (Das et al., 1981) and porcine units/mg protein. This value is higher than that brain enzyme (Asaoka and Takahashi, 1983). The reported for other mammalian species (Dillio et al., relative specific activity of glutathione S-transferase 1982; Asaoka and Takahashi, 1983; Theodore et al., in camel brain is in the order of pons > medulla 1985). The purification scheme reported in the o b l o n g a t a > thalmus > cerebrum > cerebellum > present study resulted in a homogeneous enzyme and cerebral cortex, which is very similar to that obtained enabled us to compare the camel brain glutathione for rat and porcine brain enzymes. The present study S-transferase with other mammalian brain enzymes. showed that the bulk of glutathione S-transferase Much like other transferases, the present enzyme is a activity is located in the camel brain cytosol. Similar
Rocket immunoelectrophoresis showed that the pons had the highest amount of the enzyme and the cortex had the lowest amount (Table 3), when the results were expressed as #g enzyme/mg protein. The total content of the enzyme was shown to the the highest in the cerebrum and the lowest in the medulla oblongata and pons. The biochemical estimation and the immunological quantification of glutathione S-transferase activity among the subcellular fractions of camel brain is shown in Table 4. The specific activity of the enzyme was the highest in the cytosolic fraction and the lowest in the microsomal fraction. Thus more than 88% of the glutathione S-transferase activity resided in the cytosol. The microsomal fraction contained about about 4% of the total glutathione S-transferase activity while the mitochondrial and nuclear fraction contained 3.5 and 3.9% of the activity respectively. Similarly rocket immunoelectrophoresis clearly demonstrated the abundance of the enzyme in the cytosolic fractions.
A. A. HUNAITIand Q. ASA'D
338
results were also reported for porcine brain enzyme (Asaoka and Takahashi, 1983). The data presented in this study suggests that camel brain glutathione Stransferase resembles the enzyme present in other species in terms of mol. wt and subunit composition, but differs in isozymes composition, and antigenic properties. Acknowledgements--This work was supported by a research grant from Yarmouk University. REFERENCES
Asaoka K. and Takahashi K. (1983) Purification and properties of porcine brain. J. Biochem. 94, 1191-1199. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. Biochem. 72, 242-254. Chasseaud L. F. (1979) The role of giutathione and giutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. Adv. Cancer Res. 29, 175-274. Das M., Dixit R. D., Seth P. K. and Mukhtar H. (1981) Glutathione S-transferase activity in the brain: species, sex, regional and age differences. J. Neurochem. 35, 1439-1442. Di Llio C., Polidoro G., Ardumi A. and Federici G. (1982) Glutathione S-transferases activity from guinea pig brain a comparison with hepatic multiple forms. Gen. Pharmac. 13, 485-490. Eichberg G. H. and Karnowsky M. L. (1969) Anatomy of the central nervous system. In The Structure and Function of Nervous Tissue (Edited by Bourne G. H.), VoL 3, pp. 185-213. Academic Press, New York. Habig W. H., Pabst M. J., Fleischner G., Gatmaitan Z., Arias I. M. and Jakoby W. B. (1974). The identity of
glutathione S-transferase B with Ligandin, a major binding protein of liver. Proc. natn. Acad. Sci. USA 71, 3879-3887. Hunaiti A. A. and Owais W. M. (1985) Partial purification and comparison of giutathione S-transferase from camel brain and liver. Comp. Biochem. Physiol. 81B, 251-254. Hunaiti A. A. and Abu Khalaf I. K. (1986) The distribution and comparison ofglutathione, glutathione reductase and giutathione S-transferase in various camel tissues. Comp. Biochem. Physiol. 8511, 733-737. Jakoby W. B. and Habig W. H. (1980) Glutathione transferase. In Enzymatic Basis of Detoxication (Edited by Jakoby W. B.), Vol. 2, pp. 63-94. Academic Press, New York. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage 1"4. Nature 227, 680-685. Laurell C. B. and McKay E. J. (1981) Electroimmunoassay. In Methods in Enzymology (Edited by Langone J. J. and Vunakis H. V.), Vol. 73, pp. 339-370. Academic Press, New York. Mannervik B. (1985) The isocnzymes of glutathione Stransferase. Adv. Enzymol. 57, 593-619. Norton W. (1974) Isolation of myelin from nerve tissue. In Methods in Enzymology (Edited by Jakoby B. and Wilchek M.), Vol. 46, pp. 435--443. Academic Press, New York. Ouchterlony O. (1958) Diffusion-in-gel methods for immunological analysis. In Progress in Allergy (Edited by Kalios P.), Vol. V, pp. 1-78. Kager, Basel. Polidoro G., Di Llio C., Sacchetta P., Del Boccio G. and Frederic G. (1984) Isoelectric focusing of brain cortex glutathione S-transferase activity in mammals. Evidence that polymorphism is absent in man. Int. J. Biochem. 16, 741-746. Theodore C., Singh S. V., Hong T. D. and Awasthi Y. C. (1985) Glutathione S-transferases of human brain: evidence for two immunologically distinct types of 26,500-Mr Subunits. Biochem. J. 225, 375-382.
0305-0491/89 $3.00+ 0.00 © 1989PergamonPress pie
CAMEL BRAIN GLUTATHIONE S-TRANSFERASE PURIFICATION, PROPERTIES, REGIONAL AND SUBCELLULAR DISTRIBUTION A. A. H U N A m * and Q. ASA'D Laboratory of Biochemistry and Molecular Biology, Department of BiologicalSciences, Yarmouk University,Irbid,Jordan. (Tel:0271100) (Recewed22 June 1988)
Abstract--l. Camel brain glutathione S-transferase was purified by glutathione-linked agarose affinity column and the different isozymes were separated by chromatofocusing. 2. The basic isozymes which comprise 45% of the total activity were immunologicallyindistinguishable from the near-neutral isozymes which constitute 55% of the activity. 3. Some differenceswere detectable among the basic and near-neutral isozymes in relation to substrate specificities and subunit composition. 4. Biochemical and immunological quantification of glutathione S-transferase revealed the presence of the enzyme in all camel brain regions tested and subcellular fractions. 5. The pons had the highest concentration of the enzyme and the cortex had the lowest, while more than 88% of the enzyme was present in the cytosoL
INTRODUCTION
MATERIALS
The giutathione S-transferases (EC 2.5.1.18) are a family of multifunctional proteins which catalyse the conjugation of glutathione with a broad variety of electrophilic and lipophilic compounds, many of which are toxic, mutagenic and carcinogenic (Chasseud, 1979). In mammals the transferases are all dimeric proteins of mol. wt approx. 50,000 and composed of multiple forms which are distinguished from one another by their subunit compositions and electrophoretic mobilities (Jakoby and HHabig, 1980). In guinea pig brain four major forms of the enzyme have been purified (Di Llio et aL, 1982) while three forms have been identified in human brain (Theodore et aL, 1985). Moreover, Polidoro et aL (1984) observed a remarkable difference in the nature and number of various forms of glutathione S-transferase obtained from brain cortex of cattle, sheep, mouse, guinea pig and human. In our previous work (I-Iunaiti and Owais, 1985; Hunaiti and Abu Khalaf, 1986) we demonstrated the presence of glutathione S-transferase in camel brain homogenate. However, little information is available concerning the subcellular and regional distribution of the enzyme in the brains of various animals including camel. An attempt to relate the presence of glutathione S-transferase in the brain to its role in the detoxification of various neurotoxins, implies a knowledge of the level of this enzyme in different subcellular and regional fractions of the brain. Therefore, in this paper we report on the purification of glutathione S-transferase from camel brain homogenate and the biochemical and immunological quantification of this enzyme in various subcellular and regional fractions of camel brain. *Author to whom correspondence should be addressed.
ANDMETHODS
Materials All biochemicalsused in the present study were purchased from Siama Chemical Company (USA) except for polybuffer exchangers which were from Pharmacia (Sweden)and l-chioro-2,4-dinitrobenzene and trans-4-phenyl-3-buten-2one were from Aldrich (England). Brain of l-year-old onehumped camel (Camelua dromedar/us) was obtained from a local slaughter house immediately after killing the animal and transported under ice to the laboratory. Enzyme assay The glutathione S-trausferase activity toward pnitrobenzylchioride, 1,2-epoxy-3-(p-nitrophenoxy)propane, l-chloro-2,4-dinitrobenzene, ethacrynic acid and trans4-phenyl-3-buten-2-one was determined spectrophotometrically using a Pye Unicam SP 3-400 Spectrophotometer essentially as described by Habig et ai. (1974). Enzyme activity was expressed as /zmol/min and specific activity as ~mol/min/mg protein. Protein concentrations were determined by the Bradford method (Bradford, 1976) using reagents from Bio-Rad (USA). Preparation of crude extract Fresh camel brain (316g) was washed with ice-cold distilled water and sliced into small pieces. The tissues were homogenized in 474ml of 0.1 M potassium phosphate buffer pH 7.0 containing 1.4 mM ~-mereaptoethanol using a Waring blender operating at maximum speed for 1 rain. All further steps were performed at 4°C. The homogenate was centrifuged at 37,000g for 30 min, then the supernatant was filtered through a plug of glass wool to remove floating particles. Fractionation of camel brain~ Cerebral cortex, cerebellum, cerebrum, medulla oblongata, thalrnus, hypothalmus and pons were carefully excised from the whole camel brain and crude extracts of the various regions were prepared essentially as described by Eichberg and Karnosky (1969). White matter (100g) of camel brain was used to prepare myelin fraction as described by Norton (1974). Transverse section (100 g) of
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fresh camel brain was homogenized as described above and centrifuged at 1000g for 10 vain. The pellet was suspended in (1:1.5 w/v) 0.1 M potassium phosphate buffer pH 7.2, recentrifuged, resuspended and labelled as nuclear fraction. The original supematants were further fractionated by centrifugation at 1200g for 15 rain and the resulting pellet was washed twice with the same buffer and labelled as mitocliondrial fraction. Microsomal and cytosolic fractions were obtained by further centrifugation of the supematant at 105,000g for 60rain.
Purification of glutathione S-transferase by affinity chromatography The filtrate obtained from the previous step was applied to a column (1 x 10 cm) packed with glutathione linked to epoxy activated agarose at a flow rate of 10 ml/hr after a pre-equilibration with 22 mM potassium phosphate buffer pH 7.0. The column was washed thoroughly with the same buffer containing 0.1 M NaCI until the protein absorbance at 280 nm of the effluent returned to zero. Subsequently the bound enzyme was eluted with 50mM Tris-HCl buffer, pH 9.2 containing 10 mM reduced glutathione. The fractions (2 ml each) having the highest enzymatic activity were pooled and dialysed overnight against distilled water (300 vol. two changes). The dialyzed sample was concentrated to 4 ml using an Amicon Ultra filtration cell equipped with PM 30 membrane. The concentrated enzyme was subsequently diluted with 15 volumes of double distilled water and concentrated to 4 ml.
Chromatofocusing The concentrated sample (28 mg) was applied to a chromatofocusing column (0.9 x 40 cm) packed with polybuffer exchanger 94 which had been equilibrated with 15 bed volumes of the starting buffer (0.025 M ethanolamine-HCl pH 9.4). The different isozymes were ¢luted with polybuffer 96 (diluted 1 : I0 with distilled water and adjusted to pH 6.0 with acetic acid), until the pH of eluent reached 6.0. Then a second eluent polybuffer 64 (diluted 1 : 8 and adjusted to pH 4 with HCI) was used. The flow rate was 18 ml/hr and 0.8 ml fractions were collected. Each enzymatic peak was collected and dialysed overnight against 0.1 M potassium phosphate buffer 7.2. Polybuffer was removed from all samples by affinity chromatography as described above.
Electrophoresis Analytical gel electrophoresis of the purified enzyme (20-30 #g) was carded out at 4°C in 15% SDSpolyacrylamide gel as described by Laemmli (1970).
Antibody production Antibodies against affinity purified glutathione Stransferases and isoenzyme C: (pI 7.9) and C6 (pI 7.2) were raised in three different rabbits. In each case about 400 #g protein was thoroughly mixed with Frennd's complete adjuvant (1 : 1 v/v) and the resulting emulsion (0.8 ml) was
injected intramuscularly into three different sites of one of the large hind thighs of the rabbit. Two weeks later a similar injection in Freund's incomplete adjuvant was made. After 3 weeks, the rabbits were bled and the antisera were collected.
Immunodiffusion Ouchteriony double diffusion analysis was performed on microscopic slides with 1.5% agarose in 75 mM barbital buffer pH 8.6 (Ouchterlony, 1958). After 2448 hr the nonagglutinated proteins were removed by copious washing with 0.15MNaC1 and the immunopr~pitin lines were visualized by staining with Coomassie Brilliant Blue R-250.
Rocket immunoelectrophoresis Rocket immunoelectrophoresis was performed on (7.5 x 5cm) preheated glass plates loading with 1.5% agarose which was prepared in 75raM barbital buffer pHS.5, 5mM EDTA, 0.02% sodium azide and 0.9% NaC1 essentially as described (Laurell and McKay, 1981). Electrophoresis was performed at 4°C and 10 V/cm using BRL horizontal electrophoresis. Rockets were usually formed after 4-6 hr. RESULTS
Purification of glutathione S-transferase from camel brain Crude extracts prepared from camel brain contained considerable amounts of glutathione Stransferase activity about 20 units/gram wet wt. The specific activity towards the most c o m m o n l y used substrate l,chloro-2,4-dinitrobenzene ranged from 1.8 to 2.3 units/rag protein. Application o f the 37,000g supematant onto a glutathione agaros¢ affinity column resulted in retaining all the enzymatic activity but not the bulk o f the protein. Elution of the column with 10 m M glutathione recovered more than 62% of the applied activity (Table l) in a single sharp symmetrical peak o f activity. It was estimated that glutathione Stransferase activity represents about 1.4% o f the total extractable protein of camel brain. When the glutathione S-transferase obtained from the affinity column was applied to the chromatofocusing column, the enzyme was resolved into at least eight distinct peaks ( F i g . 1). The five basic isoenzymes which were eluted between p H 8.0 and p H 7.4 accounted for about 45% of the total enzymatic activity towards 1-chloro-2,4-dinitrobenzene. The major basic isoenzyme with pI 7.9 constituted about 70% of the total basic isoenzymes. The three near-neutral isoenzymes which were eluted between p H 7 . 3
Table 1. Purification of glutathione S-transferase from camel brain Specific Total Activity activity Purification Yield Purification step protein (total units) (units/mg) (fold) (%) Crude extract 3316.8 6168 1.859 1.0 100 Affinity chromatography 28.0 3840 137.0 73.6 62 Chromatofocusing Peak C1 (pI 7.95) 0.04 1.1 27.5 14.8 0.0178 Peak C2 (pl 7.85) 1.05 158.0 150.0 80.6 2.56 Peak C3 (pI 7.8) 0.61 49.0 78.0 42.1 0.79 Peak C4 (pl 7.6) 0.133 19.0 140.0 75.3 0.308 Peak C5 (pI 7.4) 0.0275 6.0 54.5 29.3 0.097 Peak C6 (pI 7.3) 0.45 83 180.0 97.0 1.345 Peak C7 (pl 7.0) 0.182 40.6 166 89.7 0.65 Peak C8 (pI 6.9) 1.5 160.0 106.0 57.2 2.5 Enzyme was purified from 316 g (wet wt) of camel brain by the methods described in Materials and Methods.
Camel glutathione S-transferase
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Fig. 1. Chromatofocusingprofile of purified giutathione S-transferase from camel brain. Experimental details are given in the text. and pH 6.9 exhibited about 55% of the enzymatic activity. No acidic isoenzymes were observed even when five different camel brains were analysed by this method.
Biochemical properties of camel brain glutathione S-transferase Figure 2 shows the SDS-polyacrylamide gel electrophoresis of the enzyme preparations obtained during the purification process. The affinity purified enzyme exhibited two protein bands at lit, 26,300 and 24,000 (Fig. 2, lane 7). The individual peaks of glutathione S-transferase obtained from chromatofocusing column were also analysed by SDSelectrophoresis. The near-neutral peaks (]6 and C7 revealed the presence of a single protein band at M, 26,300 (Fig. 2, lanes 1 and 2). The basic isozymes C3 and C, exhibited two protein bands at 26,300 and 24,000 (Fig. 2, lanes 3 and 4), while the basic Cs isozymes consisted primarily of the lower protein band M, 24,000 and faint band at M, 26,300 (Fig. 2, lane 6). Other isoenzymes were not analysed. The native mol. wt of the purified enzyme as determined by calibrated sephadex G-100 column was about 52,000. Thus the structure of camel brain glutathione S-transferase is a dimeric protein. Substrate specificities of the major camel brain glutathione S-transferase isozymes were determined and the results are shown in Table 2. The preferred substrate for the tested isozymes was 1-chloro-2,4-dinitrobenzene. Significant differences
were also observed between the basic and the near-neutral isozymes towards different substrates. The basic form had higher specific activity for 1,2-epoxy-3(p-nitropbenoxy)propane. The nearneutral isozymes but not the basic isozymes exhibited glutathione peroxide activity (Table 2).
Stability The freezing and thawing process of the purified enzyme caused a loss of 10-15% of the activity, while incubation of the enzyme at 60°C for 4 rain resulted in 90% loss of the activity. Storage of the purified enzyme for 1 yr at -20°C caused loss of about 20% of the initial activity with concurrent appearance of four protein bands on SDS-gel electrophoresis. Immunological properties of camel brain glutathione S-transferase Immunotitration showed that when antibodies raised against the major C2(pI 7.9), Cr(pI 7.2) and affinity purified pool glutathion¢ S-transferase were incubated with the respective enzyme, the enzymatic activity towards l-chloro-2,4-dinitrobenzene was inhibited in a concentration-dependent manner. Ouchterlony double diffusion analysis showed that antibodies raised against camel brain glutathione S-transferase cross-reacted only with camel brain homogenate, but not with homogenates prepared from rabbit, mouse, rat, guinea pig, sheep and cow brains (Fig. 3). Similarly the antibodies were also
Table 2. Substrate spccificities of different camel brain glutathione S-transferase isozymes Glutathione S transferase activity (~mol/min/mg) Major basic isozyme Substrate Affinity pool (pI 7.9) 1-Chloro-2,4-dinitrobenzene 65.6 58.14 Nitrobenzyl chloride 8.87 0.75 Ethacrynic acid 5.68 7.1 1,2-Epoxy-p-nitrophenoxy propane 2.3 1.14 Trans-4-phenyl-3-buten-2-one 0.0015 ND Cumene hydroperoxide* ND ND Experimental details are indicated in the text. ND, not detected. *Glutathione peroxidase activity.
Major near neutral isozyme (pI 6.9) 91.6 3.31 0.84 6.3 ND 0.05
336
A.A. HUNAITIand Q. ASA'D
Fig. 2. SDS-polyacrylamide gel electrophoresis (15%) of camel brain glutathione S-transferases. Lane 1 C6 (pI 7.2) isozyme, lane 2 C7 (pI 6.9) isozyme, lane 3 (pI 7.3) isozyme, lane 4 CA (pI 7.6) isozyme, lane 5 C6 isozyme, lane 6 C5 (pI 7.4) isozyme, lane 7 affinity pool enzyme. Lane 8 protein standards {bovine serum albumin 66,000, ovalbumin 45,000, glyceraldehyde 3-phosphate dehydrogenase 36,000, carbonic anbydrase 29,000, trypsinogen 24,000, trypsin inhibitor 20, 100 and/~-lactoalbumin 14,400). In each case about 10/~g proteins were loaded. shown to react with crude extracts prepared from various camel organs such as lung, heart, retina, kidney and liver (data not shown).
Regional and subcellular distribution of the enzyme When the various regions of camel brain were carefully separated and the glutathione S-transferase activity was measured in each region, some
differences in the total and specific activity were observed (Table 3). The highest specific activity was found in the pons while the lowest specific activity was associated with the cortex. The total enzyme activity was highest in the cerebrum and lowest in the medulla oblongata. Moreover, the distribution of glutathione S-transferase in different regions of camel brain was quantified by an immunological technique.
Fig. 3. Ouchterlony double-diffusion analysis of glutathione S-transferases in brain cytosol of several mammalian species. Centre wells contain 0.25 mg of antibody raised against atfmity purified camel enzyme. Outer wells contain 0.5 mg of brain cytosols of C, camel', RAB, rabbit; M, mouse; RAT; GP, guinea pig; S, sheep, Cow.
Camel glutathione S-transferase
337
Table 3. Biochemicaland immunologicalquantitation of giutathione S-transferasein different regions of camel brain* Total giutathione Total activity Specificactivity S-transferase Brain region 0zmol/min) ~mol/min/mg) 0~g/mg protein)$ Cerebellum 13,158 3.62 t.25 Cortex 513 1.43 0.75 Cerebrum 3120 3.42 1.38 Medulla oblongata 95 3.85 3.2 Thalmus 680 3.65 2.3 Hypothalmus 975 3.32 2.0 Pons 385 6.64 3.5 Remainderi" 3540 3.58 1.3 *Prepared from 635 g of camel brain tissue. ")'Remainderrefers to other parts of the brain not already specifiedin the table. :~Determinedby rocket immunoelectrophoresis. Table 4. Subcellulardistributionof glutathione S-transferas¢in camel brain* Specific Total .giutathione Activity activity S-transferase ~mol/min) (/~mol/min/mg) ~g/mg protein)t Fraction Nuclear fraction 29.5 0.588 0.4 Mitochondrial fraction 39.7 0.629 0.32 Mierosomalfraction 45.9 0.316 0.068 Cytosol 92.5 61.5 12.5 Myelin sheath 39.37 1.89 3.84 *Prepared from 300 g of camel brain tissue. tDetermined by rocket electrophoresis. dimeric protein with a native mol. wt of 52,000. The subunit composition and molecular weights are in close agreement with those reported for other species (Jakoby and Habig, 1980; Theodore et al., 1985; Mannervik, 1985). Isoelectric fractionation of camel brain giutathione S-transferase resulted in a distinguished isozyme pattern with basic forms comprising about 45% of the total activity and a near-neutral comprising about 55% of the activity and complete absence of acidic forms. This is quite different from the isozymes pattern of human brain in which about 85% of the activity was due to acidic forms (Theodore et al., 1985). It appears that the antigenic determinants present in camel brain glutathione S-transferase are distinct from those present in brain cytosols of mice, guinea pigs, rabbits, cows and sheep. Furthermore, the immunological studies do suggest a close relationship between the different forms of glutathione S-transferase in camel brain tissue and the various forms of the enzyme in camel lung, retina, heart and liver. DISCUSSION To find out whether the whole brain or a specific Our study clearly demonstrates that camel brain part of it engaged in the detoxification of endocontains considerable amounts of glutathione S- geneous and/or exogenous eleetrophilic toxicants, transferase. The content of this enzyme in camel biochemical and immunological quantification of brain is about 20 units/g wet wt. This level is similar the enzyme were employed. The results of such an to the previously reported value for human brain investigated clearly showed the distribution of the which was about 12 units/g wet wt (Theodore et al., enzyme in various brain regions. These findings 1985). The specific activity of the enzyme in crude are in close agreement with those reported for rat extracts from camel brain ranges from 1.8 to 2.3 brain enzyme (Das et al., 1981) and porcine units/mg protein. This value is higher than that brain enzyme (Asaoka and Takahashi, 1983). The reported for other mammalian species (Dillio et al., relative specific activity of glutathione S-transferase 1982; Asaoka and Takahashi, 1983; Theodore et al., in camel brain is in the order of pons > medulla 1985). The purification scheme reported in the o b l o n g a t a > thalmus > cerebrum > cerebellum > present study resulted in a homogeneous enzyme and cerebral cortex, which is very similar to that obtained enabled us to compare the camel brain glutathione for rat and porcine brain enzymes. The present study S-transferase with other mammalian brain enzymes. showed that the bulk of glutathione S-transferase Much like other transferases, the present enzyme is a activity is located in the camel brain cytosol. Similar
Rocket immunoelectrophoresis showed that the pons had the highest amount of the enzyme and the cortex had the lowest amount (Table 3), when the results were expressed as #g enzyme/mg protein. The total content of the enzyme was shown to the the highest in the cerebrum and the lowest in the medulla oblongata and pons. The biochemical estimation and the immunological quantification of glutathione S-transferase activity among the subcellular fractions of camel brain is shown in Table 4. The specific activity of the enzyme was the highest in the cytosolic fraction and the lowest in the microsomal fraction. Thus more than 88% of the glutathione S-transferase activity resided in the cytosol. The microsomal fraction contained about about 4% of the total glutathione S-transferase activity while the mitochondrial and nuclear fraction contained 3.5 and 3.9% of the activity respectively. Similarly rocket immunoelectrophoresis clearly demonstrated the abundance of the enzyme in the cytosolic fractions.
A. A. HUNAITIand Q. ASA'D
338
results were also reported for porcine brain enzyme (Asaoka and Takahashi, 1983). The data presented in this study suggests that camel brain glutathione Stransferase resembles the enzyme present in other species in terms of mol. wt and subunit composition, but differs in isozymes composition, and antigenic properties. Acknowledgements--This work was supported by a research grant from Yarmouk University. REFERENCES
Asaoka K. and Takahashi K. (1983) Purification and properties of porcine brain. J. Biochem. 94, 1191-1199. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. Biochem. 72, 242-254. Chasseaud L. F. (1979) The role of giutathione and giutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. Adv. Cancer Res. 29, 175-274. Das M., Dixit R. D., Seth P. K. and Mukhtar H. (1981) Glutathione S-transferase activity in the brain: species, sex, regional and age differences. J. Neurochem. 35, 1439-1442. Di Llio C., Polidoro G., Ardumi A. and Federici G. (1982) Glutathione S-transferases activity from guinea pig brain a comparison with hepatic multiple forms. Gen. Pharmac. 13, 485-490. Eichberg G. H. and Karnowsky M. L. (1969) Anatomy of the central nervous system. In The Structure and Function of Nervous Tissue (Edited by Bourne G. H.), VoL 3, pp. 185-213. Academic Press, New York. Habig W. H., Pabst M. J., Fleischner G., Gatmaitan Z., Arias I. M. and Jakoby W. B. (1974). The identity of
glutathione S-transferase B with Ligandin, a major binding protein of liver. Proc. natn. Acad. Sci. USA 71, 3879-3887. Hunaiti A. A. and Owais W. M. (1985) Partial purification and comparison of giutathione S-transferase from camel brain and liver. Comp. Biochem. Physiol. 81B, 251-254. Hunaiti A. A. and Abu Khalaf I. K. (1986) The distribution and comparison ofglutathione, glutathione reductase and giutathione S-transferase in various camel tissues. Comp. Biochem. Physiol. 8511, 733-737. Jakoby W. B. and Habig W. H. (1980) Glutathione transferase. In Enzymatic Basis of Detoxication (Edited by Jakoby W. B.), Vol. 2, pp. 63-94. Academic Press, New York. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage 1"4. Nature 227, 680-685. Laurell C. B. and McKay E. J. (1981) Electroimmunoassay. In Methods in Enzymology (Edited by Langone J. J. and Vunakis H. V.), Vol. 73, pp. 339-370. Academic Press, New York. Mannervik B. (1985) The isocnzymes of glutathione Stransferase. Adv. Enzymol. 57, 593-619. Norton W. (1974) Isolation of myelin from nerve tissue. In Methods in Enzymology (Edited by Jakoby B. and Wilchek M.), Vol. 46, pp. 435--443. Academic Press, New York. Ouchterlony O. (1958) Diffusion-in-gel methods for immunological analysis. In Progress in Allergy (Edited by Kalios P.), Vol. V, pp. 1-78. Kager, Basel. Polidoro G., Di Llio C., Sacchetta P., Del Boccio G. and Frederic G. (1984) Isoelectric focusing of brain cortex glutathione S-transferase activity in mammals. Evidence that polymorphism is absent in man. Int. J. Biochem. 16, 741-746. Theodore C., Singh S. V., Hong T. D. and Awasthi Y. C. (1985) Glutathione S-transferases of human brain: evidence for two immunologically distinct types of 26,500-Mr Subunits. Biochem. J. 225, 375-382.