Analysis of glutathione S-transferase from human liver by isoelectric focusing in a urea minigel system

ANALYTICAL

BIOCHEMISTRY

1%,255-261

(1991)

Analysis of Glutathione S-Transferase from Human Liver by Isoelectric Focusing in a Urea Minigel System Kathleen

A. Killickl

Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, Boston, Massachusetts 02115

Received

November

Glutathione transferases (GST)’ are multifunctional enzymes present in a variety of tissues from many different species of organisms. Reactions catalyzed by these enzymes include: (i) conjugation of reduced glutathione (GSH) to numerous drugs, carcinogens, and endogenous steroids; (ii) detoxification of lipid and nucleic acid hydroperoxides; and (iii) noncovalent binding of

1 Present address: Tuft’s University, School of Medicine, Department of Physiology, 136 Harrison Ave., Boston, MA 02111. ’ Abbreviations used: GST, glutathione S-transferase; IEF, isoelectric focusing; CDNB, l-chloro-2,4-dinitrobenzene; CHAPS, 3-[(3cholamidopropyl)dimethyl ammoniol-1-propanesulfonate; TEMED, N,N,N’,N’-tetramethylenediamine; EIA, enzyme immunoassay; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SSB, sample solubilization buffer; SOB, sample overlay buffer. Copyright All

rights

$3.00 1991 by of reproduction

0

Cancer Institute,

26,199O

A method for the rapid analysis of isozyme subunits of glutathione transferase (GST) from human liver is described. Following purification of enzyme protein to electrophoretic homogeneity on columns of GSH-agarose, pooled transferase fractions were concentrated by ultrafiltration and subjected to further fractionation and analysis by urea-isoelectric focusing in minigels using a Hoefer Mighty Small II electrophoresis system. These methods combined with immunoblotting techniques permitted the resolution, detection, and eventual analysis of up to six different subunits of the o! isozyme of human GST and at least three to four different forms of the r isozyme of the transferase. The rapidity, accuracy, and sensitivity of the methodology may prove useful to the analysis and quantification of GST subunits in biopsies of malignant human tissue and to the development of effective chemotherapeutic regimens. 0 1991 Academic Press, Inc.

0003.2697/91

and the Dana-Farber

nonsubstrate hydrophobic ligands, e.g., heme and bilirubin (i.e., ligand function) (1). Structurally diverse, the transferases are dimeric proteins having a wide range of isoelectric points (i.e., pI 4.7-9.8). Based on these differences in pls, GSTs are conveniently divided into three major classes: (i) CY(basic); (ii) p (near neutral); and (iii) ?r (acidic). Each of these classes is representative of a separate multigene family. Subunit molecular weights are heterogeneous and range from 23,000 (a) to 25,000 (CY)to 26,700 (FL). Additionally, many isoforms have been reported to exist within each class. While some of these enzymes arise as a consequence of post-translational modification reactions such as glycosylation (a isoforms) and methylation reactions (h), the basis for the presence of the majority of the remaining isozymes (i.e., a) is unknown. Resolution and subsequent purification of the different forms of human liver GST has been achieved using a variety of different chromatographic techniques, e.g., affinity chromatography, fast protein liquid chromatography and reverse-phase high-performance liquid chromatography (2-4). Vander Jag-t et al. (2) using a combination of affinity chromatography and chromatofocusing techniques identified 13 different forms of native GST in human liver. The majority of these enzymes had pIs ranging from 7.35 to 8.87. While most human livers exhibit marked heterogeneity relative to GST isozyme content, only about 50-60s of the human population contain the p form in their livers. The presence and degree of microheterogeneity among the ?r isozymes is variable. Alterations in GSTs (i.e., protein level, isozyme profile, and catalytic specificity) are often associated with the development of tumor cell resistance either in vitro or in viva during a course of chemotherapy (5-8). Although there are several mechanisms whereby changes in GST activity in vim might contribute to drug resis255

Academic in any

Press, Inc. form

reserved.

256

KATHLEEN

tance, metabolism of antineoplastic agents by the GSTs has not been extensively studied. Even where GST class specificity (i.e., a) has been observed in alkylating agent metabolism (i.e., melphalan) little is known concerning the metabolic roles played by individual GSTs. Research in this laboratory has recently been concerned with determining the kinetics of melphalan conjugation by GSTs purified from human liver. One goal of this research is to determine whether there are striking kinetic differences in the metabolism of this drug and many others by the widely heterogeneous o-class of GSTs. If such a correlation does exist, it should be possible to correlate GST isozyme profiles from individual livers with conjugation activity toward specific anticancer drugs (i.e., alkylating agents) and thus to define which drugs would be the least susceptible to in vivo inactivation (i.e., metabolism) by human liver GSTs. Hence, the most appropriate drug for use during therapy as well as during the onset of acquired drug resistance could be prescribed. During the course of kinetic studies on melphalan metabolism by GSTs in vitro, methods were developed to isolate, purify, and subsequently resolve subunits of individual human liver GSTs using urea-IEF in a minigel slab system. The procedures to be described are of significance to clinical research, since the methodology is well suited to the rapid evaluation of the GST subunit composition of numerous tumor biopsy samples. MATERIALS

GSH-agarose, l-chloro-2,4dinitrobenezene (CDNB), bovine serum albumin, GSH, trizma base, glycine, glycerol, 2-mercaptoethanol, CHAPS detergent, ampholytes, sulfosalicyclic acid, nitro blue tetrazolium, sucrose, 5-bromo-4-chloro-3-indolyl phosphate, dimethylformamide, I-arginine, and l-lysine were purchased from Sigma Chemical Co. Acrylamide, N,N’-methylenebis(acrylamide), sodium dodecyl sulfate, NJVJV’JVtetramethylenediamine (TEMED), ammonium persulfate, Coomassie brillant blue R-250 and G-250, and EIA-grade affinity-purified goat anti-rabbit IgG alkaline phosphatase conjugate, and gold enhancement kits were obtained from Bio-Rad. Urea was obtained from Bethesda Research Labs, and phosphoric acid from Fluka Chemical Co. Nitrocellulose and Immobilon polyvinylidene difluoride membranes were obtained from Hoefer and Millipore, respectively. GST antibodies specific for cy,T, and p isozymes were purchased from Med Labs (Dublin Ireland). METHODS

Assay of glutathione transferase activity. Glutathione S-transferase activity was measured at 30°C in 100 mM potassium phosphate buffer (pH 6.5) that contained 2.5 mM glutathione and 1 mM CDNB (9). The

A. KILLICK

reaction was initiated by the addition of CDNB. Initial velocities were determined spectrophotometrically by measuring the change in absorbance at 340 nm in a Beckman DU spectrophotometer. The rate of the nonenzymatically mediated conjugation reaction was determined by measuring the reaction rate in the absence of enzyme. This value was then subtracted from the rate obtained in the presence of catalytically active glutathione transferase. One unit of enzymatic activity is defined as that amount of enzyme that catalyzes the formation of 1 pmol of S-2,4,dinitrophenylglutathione per minute at 30°C with the above assay conditions. The specific activity is expressed as units per milligram of protein. Protein assay. Protein content was measured according to the methods of Lowry et al. (10) or the Bradford procedure (11) using bovine serum albumin as the standard. Purification of glutathione transferase from human liver. Postmicrosomal supernatant solutions prepared from homogenized human liver sections were exhaustively dialyzed at 4°C against several changes of phosphate-buffered saline (pH 7.4) solution. Following dialysis, the solution was clarified by centrifugation at 33,000g (4°C 20 min). Recovered glutathione transferase was then subjected to affinity chromatography (2) at 4°C on a column (30-40 cm) of GSH-agarose. After the column was thoroughly equilibrated in PBS solution, aliquots of dialyzed liver supernatant solution were applied to the column at a flow rate of 10 ml/h. Upon completion of sample application, the column resin was washed with 5 bed vol of PBS solution at a flow rate of 30 ml/h. This step removes protein that does not specifically bind to the affinity agarose. The column was washed with 5 bed vol of 50 mM Tris buffer (pH 9.6). Enzymatic activity eluted during this step comprises that fraction of glutathione transferase activity referred to as the low affinity fraction. The column was washed with 50 mM Tris buffer (pH 9.6) that contained 5 InM glutathione. Transferase activity eluted at this step is referred to as high affinity enzyme. Low and high affinity glutathione transferase fractions were concentrated by ultrafiltration in an Amicon cell at 4°C using a PM10 membrane filter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was carried out according to the procedures of Laemmli (12). Total acrylamide concentrations in separating and stacking gels were 12.5 and 4%, respectively. % C for both gel layers was 2.7. Electrophoresis was performed either in glass rods or in vertical slabs. For rod electrophoresis a Canalto apparatus was used. For slab gels, the SE 250 Mighty Small II vertical slab gel electrophoresis unit from Hoefer was used. Rod gels were electrophoresed at a constant current of 2 mA/gel. Slab gels were electro-

GLUTATHIONE

phoresed at 60 V constant voltage until the sample had migrated into the stacking gel. The voltage was then increased to and maintained at 120 V until completion of the electrophoretic run. Staining of gels following SDS-PAGE. Following electrophoresis, gels were stained with a 0.25% (w/v) solution of Coomassie brilliant blue R-250 in 40% methanol/10% acetic acid. Gels were stained overnight at room temperature and were then destained in a solution of 20% methanol/lo% acetic acid. Urea-isoelectric focusing in minigels. Urea-IEF gels (7 cm) were cast in a Hoefer Mighty Small Multiple Gel Caster at a thickness of 1.5 mm. Gels to be used for resolution of (Y GST subunits consisted of the following additives in a total volume of 60 ml: 10 ml stock acrylamide solution (28.4% (w/v) acrylamide, 1.6% (w/v) bis(acrylamide), 1.2 g CHAPS detergent, 2.40 ml pH 6.8 ampholytes, and 0.60 ml pH 3-10 ampholytes. Urea gels to be used for p and a GST isozyme subunit resolution consisted of the above receipe modified such that the ampholyte composition consisted of 2.4 ml pH 4-6.5 plus 0.60 ml pH 3-10 ampholytes per 60-ml final volume. This solution was degassed and 135 ~1 ammonium persulfate (10% (w/v)) and 68 ~1 TEMED were added. After the gel was cast, either a 5- or a lo-well comb was inserted before polymerization was allowed to proceed. After polymerization was completed, the combs were removed and the wells were rinsed with distilled water. The wells and upper chamber were filled with anode solution (25 mM phosphoric acid) and the lower chamber was filled with 50 mM NaOH (cathode solution). Electrophoresis was conducted at room temperature for 30 min at 150 V, constant voltage, and then at 200 V for 2.5 h (3). After focusing was completed, the gel was removed and was either stained with Coomassie brillant blue R-250 or was subjected to immunoblotting. Urea-isoelectric focusing in maxigels. Urea-IEF gels were cast at a thickness of 0.75 mm in the Hoefer SE600 vertical slab gel electrophoresis apparatus. Urea gels consisted of the following additives in a total volume of 50 ml: 8.34 ml stock acrylamide solution (28.4% (w/v) acrylamide, 1.6% (w/v) bis(acrylamide)), 1.0 g CHAPS, 1.88 ml pH 3-10 ampholytes, 0.63 ml pH 6-8 ampholytes, and 24 g urea. After degassing the solution, 113 ~1 ammonium persulfate (10% (w/v)) and 57 ~1 TEMED were added. After the gel was cast, a Teflon comb was inserted before polymerization began. After polymerization was completed, the combs were removed and the wells were rinsed with distilled water. The wells and upper chamber were filled with anode solution (25 mM phosphoric acid) and the lower chamber was filled with cathode solution (25 mM arginine, 20 mM lysine, 120 ml ethylene diamine per liter final volume). Prefocusing was conducted at room temperature for 30 min at 200 V, 30 min at 400 V, and 800 V,

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257

for 30 min. Focusing was then conducted at 800 V for 3.5 to 4.0 h. Sample preparation for IEF. Sample solubilization buffer (SSB) was prepared as 1.5-fold concentrated stocks and consisted of: 0.15 g CHAPS, 3.0 g urea, 188 ~1 pH 3-10 ampholytes, and 62.5 ~1 pH 6-8 ampholytes. The pH was adjusted to 4.5 with HCl and the final volume was adjusted to 4.75 ml with distilled water. Aliquots of 0.95 ml were stored at -20°C. Before use, SSB solutions (i.e., 0.95 ml) were thawed and 50 ~12-mercaptoethanol (14 M) was added per 0.95-ml aliquot. For focusing, 1 vol of protein sample was mixed with 2 vol of SSB. Aliquots were then added to the gel wells. Applied samples (50-75 ~1 per lane) were overlayed with sample overlay buffer (SOB). SOB consisted of: 95 ~1 pH 3-10 ampholytes, 30 ~1 pH 6-8 ampholytes, 0.15 mg CHAPS, 1.65 g urea per final volume of 4.75 ml (pH adjusted to 4.3). Aliquots of 0.95 ml were stored at -20°C. Prior to use, 50 ~1 of 2-mercaptoethanol (14 M) was added per 0.95-ml aliquot. Prior to electrophoresis, the electrodes were connected so that the polarity was reversed. Staining ofgels following denaturing IEF in urea. Following electrophoresis, the gels were removed and placed into a solution of 4.4% (w/v) sulfosalicylic acid that contained 17.7% trichloroacetic acid. The gels were soaked in this solution with shaking overnight at room temperature and were then transferred to a solution of Coomassie brilliant blue (G-250) (0.04% (w/v)) in 3% perchloric acid and incubated for 1 h at 60°C. The gels were then destained in a solution consisting of 5% acetic acid, 6.65% ethanol, and 10% ethyl acetate. For further sensitivity, destained gels were restained in a solution of 0.25% (w/v) Coomassie brilliant blue R-250 in 45% methanol/lo% acetic acid and then destained in 20% methanol/lO% acetic acid. Western blot analysis. Purified transferase preparations were subjected to electrophoresis on native gels, SDS-polyacrylamide gels (12.5% standard Laemilli gels), and urea-IEF gels. Electrophoretic transfer to sheets of either nitrocellulose (Vanguard) or Immobilon (Waters Corp.) was carried out overnight in a Hoefer transphor apparatus. Prechilled buffers used for transfer (overnight at 30 V and 25°C) were: (a) SDSPAGE gels: 25 mM Tris, 192 mM glycine (pH 8.3) containing 20% methanol; (b) IEF-urea gels: 0.7% (v/v) acetic acid with 10% methanol. For the IEF-urea gels, the poles were reversed during the transfer. Following electrophoretic transfer, nonspecific protein-binding sites were blocked by incubating the blots in PBS containing 0.3% (w/v) Tween 20 (buffer I) for 1 h. The blots were then rinsed with a solution of 0.05% Tween 20 in PBS (buffer II) and subsequently transferred to sandwich bags containing the first antibody (i.e., GST specific (Y, a, or p antibody diluted l/2500 in buffer II). The bags were heat-sealed and incubated 2-4 h at 25°C with vigorous mixing on a shaker (14).

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antibody while protein associated with a minor band (Fig. 1, gel 1 upper band) reacted with p-GST-specific antibody. In only one out of seven liver cytosols examined, was the p-GST specific protein band detected (Fig. 2). The mobility of this band corresponded to a subunit M, of approximately 26,700, a value similar to that previously reported for the p subunit of GST (1). Confirmation of the identification of this specific protein subunit was subsequently obtained by the use of immunoblot analysis and antibody specific for the p isozyme of GST. The ?r isozyme (M, of 24,800) was present at low levels in the majority of liver preparations examined. Although it was not always readily detectable after staining the gels with Coomassie blue, it could be rapidly visualized after immunoblot analysis employing n-GST-specific antibody.

1

2

Urea-Isoelectric

Focusing

in Maxi- Vertical Gels

Homogeneous chromatography

samples of GST prepared on columns of GSH-agarose

by affinity were sub-

FIG. 1. SDS-PAGE of preparations of purified human liver GST. Aliquots of low and high affinity chromatography fractions corresponding to 0.5 pg of GST protein were electrophoresed on rod gels. Protein was visualized with Coomassie blue. In gel 1 (i.e., low affinity column fraction) the upper band corresponds to the p isozyme of GST while the lower band corresponds to the 01 subunit. In gel 2 (i.e., high affinity column fraction) only the a! isozyme was detected.

Blots were then washed for 5 min with solution II. Alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Bio-Rad, l/1000 dilution) was used as second antibody in incubations carried out for 2 h at 25°C. Filters were washed sequentially with buffer II, buffer I, and then buffer II again (5 miniwash). Filters were then developed in 100 mM Tris-HCl buffer (pH 8.5) that contained 100 mM NaCl, 5 mM MgCl, plus 0.33 mg/ml nitro blue tetrazolium (diluted from a 50 mg/ml stock in 70% dimethylformamide and 0.165 mg/ ml 5-bromo-4-chloro-3-indolyl phosphate. Reactions were terminated with 20 mM Tris-HCl (pH 8.0) buffer that contained 5 mM EDTA (14). RESULTS

AND

DISCUSSION

Affinity Chromatography and SDS-PAGE Glutathione transferase in postmicrosomal supernatant solutions of human liver was purified to homogeneity using affinity chromatography on columns of GSHagarose. Recovery of transferase activity from all liver samples examined was excellent (i.e., SO-100%). SDSPAGE analysis of aliquots of low and high affinity column fractions indicated the presence of a major protein band detectable after Coomassie blue staining of the gel. The mobility of this band corresponded to a subunit molecular size of 25,000. Protein associated with this major band (Fig. 1, gel 2) reacted with a-GST-specific

FIG. 2. Urea-IEF of crude preparations of GST from Postmicrosomal supernatant solutions prepared from human livers (i.e., gel lanes 1 to 6) and corresponding protein were analyzed on vertical slab urea-IEF gels in 600 electrophoresis apparatus. Protein subunits were nitrocellulose blots after reaction with r-isozyme-specific previously described under Methods.

human liver. six different to 132 ag of a Hoefer SE visualized on antibody as

GLUTATHIONE

jetted to isoelectric focusing under denaturing conditions. Gels (0.75 mm thick) containing 8 M urea and 2% CHAPS were poured and were subsequently electrophoresed in the Hoefer SE 600 vertical slab gel electrophoresis apparatus as previously described under Methods. Both Coomassie blue staining and immunoblot analysis of the gels following isoelectric focusing indicated the presence of multiple subunits of the LYisozyme of GST in several preparations of homogeneous GST analyzed. Staining of proteins transferred to nitrocellulose with the gold enhancement procedure confirmed this heterogeneity. Moreover, in samples of unfractionated cytosol, several protein bands were detected that reacted with p-GST-specific antibody (Fig. 2). Unfortunately, major disadvantages to the use of these procedures exist: (i) length of time for a single electrophoretic run; (ii) fragility of gels, making them extremely difficult to stain and manipulate during Coomassie blue or gold staining or during the preparation of Western blots; and (iii) expense. For these reasons, attempts were made to adapt these procedures to use with a Hoefer minigel system (13). Urea-Isoelectric Focusing in the Hoefer Minigel System In the adaptation of the maxigel urea-IEF system for use with the Hoefer SE 250 Mighty Small II vertical slab gel electrophoresis system, the major changes that were made were: (i) the compositions of the electrode buffers were changed to (a) lower, 50 mM NaOH, and (b) upper, 25 mM phosphoric acid, with the poles reversed; (2) the gel thickness was increased from 0.75 to 1.5 mm; and (3) the focusing conditions were modified such that proteins were prefocused for 0.5 h at 150 V followed by focusing at 200 V for 2.5 h. Solutions were prepared, gels were cast and samples were focused all on the same day. After degassing the solutions, polymerization was initiated by the addition of TEMED and ammonium persulfate and either two or four gels were cast in either the Hoefer SE 215 or SE 275 Mighty Small gel caster. All electrophoretic runs were performed at room temperature with electrode solutions previously chilled to 4°C prior to the run. The major differences in gel composition between the methods described in this paper and those of Robertson et al. (13) are summarized in Table 1. For resolution of GST isozyme subunits, glycerol and Triton X-100 were omitted. As detailed above with the maxi-vertical system, CHAPS was routinely added to a final concentration of 2%. Inclusion of this detergent in the urea gels resulted in the formation of sharp, compact protein subunit bands. Without this additive, protein bands were still detectable although resolution was diminished by the considerable trailing associated with protein subunit migration.

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TABLE 1 Gel Compositions for the Mini-Hoefer Urea-IEF System Procedure” Additive

A

Acrylamide

5%

Glycerol

2% 2% 8M

CHAPS

Ampholytes Urea Triton X-100 Ammonium persulfate TEMED D A: method

from

0.03% 0.11% this

study.

B: methods

of Robertson

B 7.7% 15.4% 3.1% 8M 0.5% 0.03% 0.25% et al. (13).

Although the total ampholyte concentration was maintained constant at a final concentration of 2%, the composition of the different ampholyte pH ranges was varied according to the isoelectric points of the specific GST isozymes being resolved. Thus the following ampholyte compositions were used: (i) a! isozyme subunits, 1.5% pH 3-10 ampholytes plus 0.5% pH 6.8 ampholytes; (ii) ?r isozyme subunits, either 2% pH 3-10 or 1.6% pH 4-6.5 plus 0.4% pH 3-10 ampholytes; (iii) p isozyme subunits 1.6% pH 4-6.5 plus 0.4% pH 3-10 ampholytes. a Subunits Analysis of immunoblots after SDS-PAGE indicated that all samples contained at least one isozyme band reactive with a-subunit-specific antibody. Further analysis of these samples by urea-IEF indicated an even greater diversity of subunit composition. Prior to IEF, aliquots of low and high affinity column fractions from each liver were mixed together in order to simulate their relative proportions in prepared liver cytosols. As shown in the analysis presented in Fig. 3, preparations of purified GSTs, when analyzed by immunoblot analysis, contained up to 6 different forms of the (Yisozyme subunit. Since GST is a dimer, the expected theoretical range for number of native isozymes of the enzyme might be rather high. Vander Jagt (2) subjected aliquots of enzyme purified through affinity columns to chromatofocusing analysis and identified approximately 13 dimeric (Y-GSTs having pIs that ranged from 4.8 to 8.8. These observations are in agreement with those reported here. Although there were variations in the number of a-subunits from one sample to another, all livers contained the pI 8.9 isozyme as a major form of GST. P Subunits In contrast to the a-subunit, only one liver of several examined (i.e., seven) contained the ct isozyme of GST (Figs. 1 and 2). Of interest is the fact that this liver

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A. KILLICK

preparation appeared to contain two to three major p-subunit bands in addition to several other minor p bands which were present at relatively lower levels in cytosolic fractions. These results are similar to those reported by Tsuchida et al. (15) who identified five forms of the p isozyme of GST in preparations from human heart. These forms of GST are of clinical significance since they are active on nitroglycerine and the nitrosoureas as substrates and are also of prognostic value because of the statistical correlation that exists between the presence of p isozymes of GST in human lung tissue and human susceptibility to lung cancer. ?r Subunits With the exception of one liver of five examined all specimens analyzed contained the ?r isozyme of GST(Fig. 4). Excellent resolution of multiple forms of these enzymes from single liver samples was achieved by urea-IEF with pH 3-10 ampholytes (2%) followed by blotting to immobilon membranes and subsequent analysis with immunodetection methods. The microheterogeneity observed among the GST ?r isozymes within a single sample as well as that observed between different liver preparations may arise as a consequence of posttranslational modifications, e.g., glycosylation reactions. In contrast to urea-IEF, only a single form of the x isozyme was detected after either native or SDSPAGE. Although this form of GST comprises a minor percentage of total GST protein from liver, it is the predominant form in human placenta. Of clinical signifi-

FIG. 4. Resolution of multiple forms of the r isozyme of GST. Following urea-IEF of 0.49 pg of a protein mixture composed of low and high affinity forms of GST in a Hoefer Mighty Small II electrophoresis apparatus and subsequent transfer to Immobilon membranes, protein subunits were visualized after reaction with r-isozyme-specific antibody as described under Methods. The arrows indicate different forms of the x isozyme of GST.

cance is the observation that circulating levels of serum ?r transferase protein increase during tumor proliferation (6). CONCLUSION

iBCDEF FIG. 3. Resolution of the LYsubunits of human liver GST on ureaIEF gels. Aliquots of purified enzyme were subjected to urea-IEF on the Hoefer Mighty Small II electrophoresis apparatus as described under Methods, Approximately 0.69 c(g of total protein representing a mixture of GST protein from low and high affinity column fractions was applied per gel lane. Protein subunits were visualized on nitrocellulose blots after reaction with or-isozyme-specific antibody as previously described under Methods. Isozyme subunits are designated numerally (i.e., 1 to 6); enzyme samples from six different human livers are designated A to F, respectively.

In conclusion, methods have been described which result in the rapid analysis of GST isozymes (i.e., 6 to 8 h time period). The steps involved are: (i) tissue homogenization; (ii) ultracentrifugation of the homogenate; (iii) column affinity chromatography followed by ultrafiltration and concentration; (iv) urea-IEF in minigels; (v) protein transfer to nitrocellulose or immobilon; and finally (vi) visualization of protein subunits. With a single Hoefer Mighty Small II unit and 2, 12-well combs, 24 samples are easily processed. The sensitivity of the described procedures together with the capacity to handle numerous tissue samples makes these methods ideally well suited for clinical usage, e.g., analysis of GST isozyme compositions in tumor biopsy specimens. The methods could also perhaps be informative in deciding the course of a chemotherapeutic regimen for individual patients, once the substrate specificities and kinetics of the individual GST isozymes are known. ACKNOWLEDGMENTS The author expresses her sincere appreciation to Dr. Emil Frei for his interest and support of this project and to Dr. David J. Waxman and Dr. John Koch for their assistance in the development of several

GLUTATHIONE of the described methodologies. Many thanks are also due to the personnel of the technical services division of the Hoefer Company for always providing expert technical advice as well as scientific insight. REFERENCES 1. Mannervik, them.

B., and Danielson, U. H. (1988) CRC Crit. Reu. Bio-

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7. Ketterer, B., Meyer, D. J., and Clark, A. G. (1988) Glutathione Conjugation, (Sies, H., and Ketterer, B., Eds.), pp. 74-135, Academic Press, San Diego, CA. 8. Morrow, C. S., and Conan, K. H. (1990) CancerCells2, 15-22. 9. Habig, W. H., Pabst, M. J., and Jacoby, W. B. (1974) J. Biol. Chem. 249,7130-7139. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 11. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 12. Laemmli, U. K. (1970) Nature 227,680-685. 13. Robertson, E. F., Dannelly, H. K., Malloy, P. J., and Reves, H. C. (1987) Anal. Biochem. 167, 290-294. 14. Waxman, D. J., LeBlanc, G. A., Morrissey, J. J., Stauton, J., and Lapenson, D. J. (1988) J. Biol. Chem. 263, 11,396-11,406. 15. Tsuchida, S., Maki, T., and Sato, K. (1990) J. Biol. Chem. 265, 7150-7157.