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A method for the localization of glutathione S-transferase isozymes after starch gel electrophoresis
ANALYTICAL
BIOCHEMISTRY
A Method
105,
147- 149 (1980)
for the Localization of Glutathione S-Transferase lsozymes after Starch Gel Electrophoresis P. G. BOARD
Department
of Human Biology, John Curtin School of Medical P.O. Box 334, Canberra City 2601, Australian
Research, Australian National Capital Territory, Australia
University,
Received November 5, 1979 A new method is described by which multiple forms of glutathione S-transferase in rat and human liver can be separated by electrophoresis in starch gel and localized on the gel surface by a specific stain, utilizing I-chloro-2+dinitrobenzene and reduced glutathione as substrates. The technique permits the comparison of samples from many individuals, and allows determination of the genetic relationships between the different glutathione Stransferase isozymes.
The glutathione S-transferases (EC 2.5. 1.18) are a family of proteins which catalyze the reaction of reduced glutathione (GSH) with a wide variety of electrophilic compounds. This general reaction is the first step in the formation of mercapturic acids (1,2), a mechanism by which numerous pharmacologically active compounds are metabolized and eliminated. The glutathione S-transferases also have a high binding affinity for many nonsubstrates which may be of endogenous (e.g., bilirubin) or exogenous origin (e.g., bromsulfophthalein (3)). Glutathione S-transferases have been studied most extensively in rat and human liver (4,5) although their occurrence in other tissues and species have been described (6,7). Until now, glutathione S-transferase isozymes have been separated by ionexchange chromatography or by isoelectric focusing and the distribution of enzyme activity determined on individual fractions or gel slices. These methods have revealed the presence of multiple forms of glutathione S-transferase in human and rat liver (4,5). The lack of a suitable method for electrophoresis and the comparison of multiple samples under the same conditions has so 147
far prevented detailed study of any genetic relationship between the various glutathione S-transferase isozymes. The present paper describes a system by which the multiple forms of glutathione S-transferase can be separated by starch gel electrophoresis and specifically localized on the gel surface. MATERIALS
AND METHODS
Human liver samples were obtained at autopsy and stored at -20°C. Rat liver was obtained from freshly sacrificed Wistar rats. Liver samples were homogenized in 50 mM Tris/HCl, pH 7.4, centrifuged at 15OOg for 10 min; the supernatant fluids were applied to Whatman 3MM filter paper strips and inserted into starch gels for electrophoresis. Horizontal starch gel electrophoresis was carried out at 5°C between cooling blocks for 16 h at 4 V/cm using a Tris-EDTAborate buffer, pH 8.6. The bridge buffer was a 1:7 dilution of a stock buffer solution containing Tris 0.9 M, boric acid 0.5 M, and disodium EDTA 0.02 M. The gel buffer was a 1:lO dilution of the same stock solution. After electrophoresis the gel was sliced and the bottom half stained for glutathione 0003-2697/80/090147-03$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
148
P. G. BOARD
Origin
*Origin
FIG. 1. Anodal and cathodal migrating components of human liver glutathione S-transferase from different individuals at pH 8.6.
S-transferase activity using a two-stage procedure. Stage 1. 1 - Chloro - 2,4 - dinitrobenzene (Fluka AG), 8 mg, in 0.8 ml ethanol is mixed +
+ Origin
with 14 mg of reduced glutathione (Sigma Chemical Co.) and dissolved in 20 ml of 0.1 M potassium phosphate, pH 6.5. The solution is used for saturating a filter paper sheet which is overlayed on the cut surface of the gel and incubated at 37°C for 40 min. Stage 2. The filter paper from Stage 1 is removed and the gel is overlayed with a developing solution prepared by combining 30 ml of iodine solution (0.9 ml of 1% I, in KI diluted in 30 ml H,O) with an equal volume of 2% molten agar. The agar sets on the gel surface and an intense blue starch-iodine color appears immediately in areas where GSH has been conjugated to l-chloro-2,4dinitrobenzene by the action of glutathione S-transferase. RESULTS AND DISCUSSION
FIG. 2. Multiple anodal and cathodal migrating components of rat liver glutathione S-transferase at pH 8.6.
The technique described here is a modification of a procedure previously presented for the detection of glyoxalase I isozymes after starch gel electrophoresis (8). The glutathione S-transferase reaction between GSH and 1-chloro-2,4-dinitrobenzene is apparently specific (9) and our experiments have shown that there is no cross-reaction
GLUTATHIONE
S-TRANSFERASE
with glyoxalase I. If 1-chloro-2,4-dinitrobenzene is omitted from the Stage 1 reaction mixture, no zones of reaction can be observed. Figure 1 shows the electrophoretic separation of human liver glutathione Stransferase at pH 8.6. There are several components that migrate toward both the anode and the cathode. The anodal and cathodal components can both be observed on the same gel if the origin is placed in the center. However, greater resolution of either anodal or cathodal migrating isozymes can be achieved separately if the origin is placed closer to either pole. The results with human liver show considerable heterogeneity in the number and electrophoretic mobility of glutathione S-transferase isozymes between individuals. Examples of this variation are shown in Fig. 1. This variation appears to be under genetic control and will be reported in detail elsewhere. Rat liver also gave rise to several isozymes (Fig. 2). Previous studies using ionexchange chromatography have indicated the presence of at least six isozymes of glutathione S-transferase, five of which are known to react with I-chloro-2,4-dinitrobenzene as a substrate (4). The method reported here provides the first opportunity for localizing glutathione S-transferase isozymes in situ after electro-
ELECTROPHORESIS
149
phoresis. The technique permits the comparison of small tissue samples from many individuals, and thus allows the comparison and genetic analysis of this important group of enzymes for the first time. ACKNOWLEDGMENTS The author thanks Ms. M. Coggan for her excellent technical assistance and Dr. Y. S. Teng for providing the liver samples.
REFERENCES 1. Boyland, E., and Chasseaud, L. F. (1969)Advan. Enzymol. 32, 172-219. 2. Chasseaud, L. F. (1976) in Glutathione: Metabolism and Function (Arias, I. M., and Jakoby, W. B., eds.), pp. 77- 114, Raven Press, New York. 3. Ketterer, B., Tipping, E., Beale, D., and Meuwissen, J. A. T. P. (1976) in Glutathione: Metabolism and Function (Arias, I. M., and Jakoby, W. B., eds.), pp. 243-257, Raven Press, New York. 4. Jakoby, W. B., Ketley, J. N., and Habig, W. H., (1976) in Glutathione: Metabolism and Function (Arias, I. M., and Jakoby, W. B., eds.), pp. 231-223 Raven Press, New York. 5. Kamisaka, K., Habig, W. H., Ketley, J. N., Arias, I. M., and Jakoby, W. B., (1975) Eur. J. Biochem. 60, 153-161. 6. Stenersen, J., Guthenberg, C., and Mannervik, B. (1979) Biochem. J. 181, 47-50. 7. Parr, C. W., Bagster, I. A., and Welch, S. G. (1977) Biochem. Genet. 15, 109-113. 8. Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) J. Biol. Chem. 249, 7130-7139.
BIOCHEMISTRY
A Method
105,
147- 149 (1980)
for the Localization of Glutathione S-Transferase lsozymes after Starch Gel Electrophoresis P. G. BOARD
Department
of Human Biology, John Curtin School of Medical P.O. Box 334, Canberra City 2601, Australian
Research, Australian National Capital Territory, Australia
University,
Received November 5, 1979 A new method is described by which multiple forms of glutathione S-transferase in rat and human liver can be separated by electrophoresis in starch gel and localized on the gel surface by a specific stain, utilizing I-chloro-2+dinitrobenzene and reduced glutathione as substrates. The technique permits the comparison of samples from many individuals, and allows determination of the genetic relationships between the different glutathione Stransferase isozymes.
The glutathione S-transferases (EC 2.5. 1.18) are a family of proteins which catalyze the reaction of reduced glutathione (GSH) with a wide variety of electrophilic compounds. This general reaction is the first step in the formation of mercapturic acids (1,2), a mechanism by which numerous pharmacologically active compounds are metabolized and eliminated. The glutathione S-transferases also have a high binding affinity for many nonsubstrates which may be of endogenous (e.g., bilirubin) or exogenous origin (e.g., bromsulfophthalein (3)). Glutathione S-transferases have been studied most extensively in rat and human liver (4,5) although their occurrence in other tissues and species have been described (6,7). Until now, glutathione S-transferase isozymes have been separated by ionexchange chromatography or by isoelectric focusing and the distribution of enzyme activity determined on individual fractions or gel slices. These methods have revealed the presence of multiple forms of glutathione S-transferase in human and rat liver (4,5). The lack of a suitable method for electrophoresis and the comparison of multiple samples under the same conditions has so 147
far prevented detailed study of any genetic relationship between the various glutathione S-transferase isozymes. The present paper describes a system by which the multiple forms of glutathione S-transferase can be separated by starch gel electrophoresis and specifically localized on the gel surface. MATERIALS
AND METHODS
Human liver samples were obtained at autopsy and stored at -20°C. Rat liver was obtained from freshly sacrificed Wistar rats. Liver samples were homogenized in 50 mM Tris/HCl, pH 7.4, centrifuged at 15OOg for 10 min; the supernatant fluids were applied to Whatman 3MM filter paper strips and inserted into starch gels for electrophoresis. Horizontal starch gel electrophoresis was carried out at 5°C between cooling blocks for 16 h at 4 V/cm using a Tris-EDTAborate buffer, pH 8.6. The bridge buffer was a 1:7 dilution of a stock buffer solution containing Tris 0.9 M, boric acid 0.5 M, and disodium EDTA 0.02 M. The gel buffer was a 1:lO dilution of the same stock solution. After electrophoresis the gel was sliced and the bottom half stained for glutathione 0003-2697/80/090147-03$02.00/O Copyright 0 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
148
P. G. BOARD
Origin
*Origin
FIG. 1. Anodal and cathodal migrating components of human liver glutathione S-transferase from different individuals at pH 8.6.
S-transferase activity using a two-stage procedure. Stage 1. 1 - Chloro - 2,4 - dinitrobenzene (Fluka AG), 8 mg, in 0.8 ml ethanol is mixed +
+ Origin
with 14 mg of reduced glutathione (Sigma Chemical Co.) and dissolved in 20 ml of 0.1 M potassium phosphate, pH 6.5. The solution is used for saturating a filter paper sheet which is overlayed on the cut surface of the gel and incubated at 37°C for 40 min. Stage 2. The filter paper from Stage 1 is removed and the gel is overlayed with a developing solution prepared by combining 30 ml of iodine solution (0.9 ml of 1% I, in KI diluted in 30 ml H,O) with an equal volume of 2% molten agar. The agar sets on the gel surface and an intense blue starch-iodine color appears immediately in areas where GSH has been conjugated to l-chloro-2,4dinitrobenzene by the action of glutathione S-transferase. RESULTS AND DISCUSSION
FIG. 2. Multiple anodal and cathodal migrating components of rat liver glutathione S-transferase at pH 8.6.
The technique described here is a modification of a procedure previously presented for the detection of glyoxalase I isozymes after starch gel electrophoresis (8). The glutathione S-transferase reaction between GSH and 1-chloro-2,4-dinitrobenzene is apparently specific (9) and our experiments have shown that there is no cross-reaction
GLUTATHIONE
S-TRANSFERASE
with glyoxalase I. If 1-chloro-2,4-dinitrobenzene is omitted from the Stage 1 reaction mixture, no zones of reaction can be observed. Figure 1 shows the electrophoretic separation of human liver glutathione Stransferase at pH 8.6. There are several components that migrate toward both the anode and the cathode. The anodal and cathodal components can both be observed on the same gel if the origin is placed in the center. However, greater resolution of either anodal or cathodal migrating isozymes can be achieved separately if the origin is placed closer to either pole. The results with human liver show considerable heterogeneity in the number and electrophoretic mobility of glutathione S-transferase isozymes between individuals. Examples of this variation are shown in Fig. 1. This variation appears to be under genetic control and will be reported in detail elsewhere. Rat liver also gave rise to several isozymes (Fig. 2). Previous studies using ionexchange chromatography have indicated the presence of at least six isozymes of glutathione S-transferase, five of which are known to react with I-chloro-2,4-dinitrobenzene as a substrate (4). The method reported here provides the first opportunity for localizing glutathione S-transferase isozymes in situ after electro-
ELECTROPHORESIS
149
phoresis. The technique permits the comparison of small tissue samples from many individuals, and thus allows the comparison and genetic analysis of this important group of enzymes for the first time. ACKNOWLEDGMENTS The author thanks Ms. M. Coggan for her excellent technical assistance and Dr. Y. S. Teng for providing the liver samples.
REFERENCES 1. Boyland, E., and Chasseaud, L. F. (1969)Advan. Enzymol. 32, 172-219. 2. Chasseaud, L. F. (1976) in Glutathione: Metabolism and Function (Arias, I. M., and Jakoby, W. B., eds.), pp. 77- 114, Raven Press, New York. 3. Ketterer, B., Tipping, E., Beale, D., and Meuwissen, J. A. T. P. (1976) in Glutathione: Metabolism and Function (Arias, I. M., and Jakoby, W. B., eds.), pp. 243-257, Raven Press, New York. 4. Jakoby, W. B., Ketley, J. N., and Habig, W. H., (1976) in Glutathione: Metabolism and Function (Arias, I. M., and Jakoby, W. B., eds.), pp. 231-223 Raven Press, New York. 5. Kamisaka, K., Habig, W. H., Ketley, J. N., Arias, I. M., and Jakoby, W. B., (1975) Eur. J. Biochem. 60, 153-161. 6. Stenersen, J., Guthenberg, C., and Mannervik, B. (1979) Biochem. J. 181, 47-50. 7. Parr, C. W., Bagster, I. A., and Welch, S. G. (1977) Biochem. Genet. 15, 109-113. 8. Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) J. Biol. Chem. 249, 7130-7139.