Comp. Biochem. PhysioL Vol. 93B, No. 2, pp. 333-338, 1989 0305-0491/89$3.00 + 0.00 Printed in Great Britain © 1989PergamonPress pie CAMEL BRAIN GLUTATHIONE S-TRANSFERASE PURIFICATION, PROPERTIES, REGIONAL AND SUBCELLULAR DISTRIBUTION A. A. HUNAm* 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 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) ob- served 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 detoxi- fication 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. MATERIALS ANDMETHODS Materials All biochemicalsused in the present study were purchased from Siama Chemical Company (USA) except for poly- buffer exchangers which were from Pharmacia (Sweden)and l-chioro-2,4-dinitrobenzene and trans-4-phenyl-3-buten-2- one were from Aldrich (England). Brain of l-year-old one- humped 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 p- nitrobenzylchioride, 1,2-epoxy-3-(p-nitrophenoxy)propane, l-chloro-2,4-dinitrobenzene, ethacrynic acid and trans- 4-phenyl-3-buten-2-one was determined spectrophoto- metrically 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 ob- longata, 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 333