Biochimica et Biophysica Acta, i 120(1992) 33-42
33
© 1992Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34138
Separation and characterization of isoforms of DT-diaphorase from rat liver cytosol Juan Segura-Aguilar a, Rudolf Kaiser b and Christina Lind a a Biochemical Toxicology, Wallenberg Laboratory, Unh'ersity o f Stockholm, Stockholm (Sweden) and h Department of Chemistry L Karolinska Institute, Stockholm (Sweden)
(Received 16 August 1991)
Keywords: Diaphorase;lsoform;Carbohydrate;DT-diaphorase;(Enzyme) Rats were treated with 3-methylcholanthrene {MC) and DT-diaphorase from liver was partially purified on an azodicoumarolSepharose 6B column and applied to an FPLC-chromatofocusing column in order to resolve isoforms. Six peaks showing significant DT-diaphorase activity were eluted from this column with a pH gradient between 7.30 to 4.80. The amino acid compositions of the two major peaks (II and Vlb) were found to be nearly identical, suggesting existence of isoforms rather than isozymes of DT-diaphorase. The isoforms of DT-diaphorase showed broad substrate specificities towards four different quinones (menadione, vitamin K-I, benzo(a)pyrene 3,6-quinone and cyclized-dopamine ortho-quinone), although quantitative differences in the specific activities were also found. All isoforms arc glycoproteins but contain different carbohydrates. Thus isoform II reacts with biotinylated lectins which are specific for N-acetylgalactosamine, mannose, fucose and galactosyl(fl-l,3)N-acetylgalactosamine, while isoform Vlb reacts only with biotinylated lectins specific for mannose and N-acetylgalaetosamine. Separation of DT-diaphorase isoforms from control rat liver cytosol using FPLC-ehromatofocusing revealed that the induction of the isoforms is not uniform, since isoform II was not found and the major isoform was composed of three peaks, whereas the major isoform of DT-diaphorase from liver cytosol of rats treated with 3-methylcholanthrene was composed of or~;: -.wo oeaks.
Introduction DT-diaphorase (NAD(P)H-quinone oxidoreductase, EC 1.6.99.2) is a flavoprotein which catalyzes the two-electron reduction of quinones, quinone epoxides and azo dyes [1-3]. This enzyme, which is induced by 3-methylcholanthrene and a large number of xenobiotics (see Refs. 4 and 5 for a review), seems to play a protective role against carcinogenicity [6], mutagenicity [7] and cytotoxicity [8] caused by quinones and their metabolic intermediates. The protective role of this enzyme seems to depend on its ability to reduce
Abbreviations: BP3,6-Q, benzo(a)pyrene 3,6-quinone; cDAoQ, cyclized doparaine ortho-quinone; DBA, dolichos biflorus agglutinin: Con A, cauavalia ensiformis agglutinin; FPLC, fast-protein liquid chromatography; IEF, isoelectric focusing; K3, raenadione; MC, 3raethylcholanthrene; MTT, 3-(4,5-diraethylthiazol-2-yl)-2,5-diphenyltetrazoliura bromide; PNA, peanut agglutinin; RCA'I, ricinus comraunis agglutininI; SBA, soybeanagglutinin;UEA 1, ulexeuropaeus agglutinin I; vit. K-l, vitamin K-I; WGA,wheat germ agglutinin. Correspondence: J. Segura-Aguilar,BiochemicalToxicology,Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm,Sweden.
quinones to hydroquinones, which are the substrates for conjugation reactions by UDP-glucuronosyl transferases and phenoisulfotranferases [9-11], and on its ability to compete with enzymes such as NADPH-cytochrome /-450 oxidoreductase, NADH-cytochrome b 5 oxidoreductase and NADH-ubiquinone oxidoreductase, which generate reactive species of oxygen when catalyzing the one-electron reduction of quinone to semiquinone [8,12]. Evidence supporting the existence of different forms of DT-diaphorase has appeared in several reports. In 1978 two different N-terminal amino acids of DT-diaphorase were reported [13]. In 1980 DT-diaphorase was reported to give rise to two distinct precipitates when antisera produced against rat liver cytosol was used in immunodifussion techniques [14]. In 1981 three different antigenic forms of isolated cytosolic as well as microsomal DT-diaphorase could be detected [15,16] whereas mitochondrial DT-diaphorase contained only two different antigenic forms [15]. In 1986 the separation of two isoforms of DT-diaphorase from murine liver cytosol, a hydrophilic and a hydrophobic form, was reported [17]. In 1987 the separation of several isoforms of DT-diaphorase on the basis of differences in their net charges was achieved [18]. The eDNA
sequences of one gene coding for DT-diaphorase [!921] from rat and human liver cytosol indicate that the various isoforms of DT-diaphorase reflect different posttranslational modifications rather than differences in primary structures. This study presents a luther characterization of DT~diaphorase isoforms and reports the separation of two additional dicoumarobbinding proteins by FPLCchromatofocusing. Materials and Methods
Chemicals Dopamine and 3-methylcholanthrene (MC) were obtained from Fluka AG (Buchs, Switzerland), Agarose C, Pharmalyte 3-10 and Polybuffer 74, low molecular weight calibration kit and isoelectric focusing calibration kit were from Pharmacia (Uppsala, Sweden). Menadione, NADH and vitamin K-l were purchased from Sigma (St Louis, MO, U.S.A.) and benzo(a)pyrene-3,6-quinone was from lIT Research Institute (Chicago, IL, U.S.A.). Lectins were from Vectors Laboratories Incorporated (Burlingame, U.S.A.) and avidin-alkaline phosphatase, 5-bromo-4-chloro-3indoyl phosphate p-toluidine salt (BCIP) and p-nitro blue tctrazolium chloride (NBT) were from Bio-Rad (California, U.S.A.). Endoglycosidase F and Endo-aN-acetylgalactosaminidase were obtained from Boehringer Mannheim (Mannheim, Germany).
Animals Male Sprague-Dawley rats of about 100 g body weight were used. The animals were kept on standard laboratory diet and starved for 24 h before death. Some animals were injected i,p. with 2 mg MC (dissolved in 0,25 m l corn oil)once daily for 3 days and decapitated 24 h after the last injection.
Isolation of DT-diaphorase isoforms by chromatofocusing Throughout this investigation liver cytosol from MC-treated and control rats has been used. Cytosol was prepared acording to Ernster et al. [22]. Cytosolic DT-diaphorase was purified as described earlier [15] by using adsorption to immobilised dicoumarol, a competitive inhibitor of the enzyme with respect to NAD(P)H. Elution of this azodicoumarol Sepharose 6B column with NADH resulted in an almost pure enzyme preparation and the recovery was about 80%. The eluate from the azodicoumarol-Sepharose 68 column was concentrated by ultrafiltration in a diaflo cell (Amicon, Lexington MA, U.S.A.) equipped with a PM-30 filter. The concentrated sample was applied to a Sephadex G-25 column equilibrated with 0.025 M imidazole-HCI, pH 7.3 + 0.25 M sucrose in order to remove NADH. This eluate was applied, at a rate of 60 m l / h, to a
mono P FP!_C chromatofocusing column (0.5 x 20 cm, Pharmacia, Sweden) equilibrated with 0.025 M imidazole-HCi, pH 7.3 + 0.25 M sucrose and eluted with polybuffer 74, pH 4.80 + 0.25 M sucrose, at 4°C.
DT-diaphorase acticity DT-diaphorase activity was measured essentially as described by Ernster et al. [1]. The standard assay system contained 50 mM Tris-HCI (pH 7.5), 0.08% Triton X-100, 500 # M NADH, 10 # M menadione and 77/zM cytochrome c. The reaction was measured spectrophotometrically by following the reduction of cytochrome c, which continuously reoxidizes menadione, at 550 nm and employing an extinction coefficient of 18.5 mM - i cm - J. When vitamin K-1 or benzo(a)pyrene 3,6-quinone was used as an electron acceptor, 0.020 mM was presented in the reaction mixture, where Triton X-100 was replaced by 0.25 M sucrose. When cDAoQ was used as an electron acceptor, DT-diaphorase activity was measured as recently reported [23], using 50 mM Tris-HCl, pH 6.5, + 0.25 M sucrose, 10 #M dopamine and 40 /~M Mn3+-pyrophosphate complex. After 2 min, 200 #M NADH was added to this reaction mixture and the reaction was started by addition of the enzyme. The reaction was followed at 475 nm and an extinction coefficient of 3058 M -~ cm- ~ was employed.
Analysis of amh~o acid composition Total amino acid composition of the isoforms of DT-diaphorase was determined with a Beckman 121M amino acid analyzer after hydrolysis for 24 h at 110°C in evacuated tubes with 6 M HCl/0.5% phenol, as described earlier [24].
Carbohydrate digestion by N- and O-glycosidases Isoforms of DT-diaphorase were incubated for 24 h at 37°C in phosphate bufter, pH 7.2 + 50 mM EDTA in the presence and in the absence of endoglycosidase F and analysed by isoelectric focusing (IEF, see below). When N-acetylgalactosaminidase was used, the isoforms were incubated in 20 mM phosphate buffer (pH 6.5) at 37°C for 24 h in the presence and in the absence of 0.1% SDS and 1% 2-mercaptoethanol. Two glycoproteins, ovalbumin and asialofetuin, were used as controls for endoglycosidase F and N-acetylgalactosaminidase activities, respectively.
Electrophoresis and isoelectric focusing (IEF) Discontinous SDS-polyacylamide gel electrophoresis was performed according Laemmli [25]. The polyacrylamide gel used for isoelectric focusing (IEF) was 1.5 mm thick and contained 5% acrylamide +0.15% bisacrylamide, 14% glycerol and 6.3% Pharmalyte 3-10. The gel was prefocused for 60 min at 7 W and focused at 7 W until 2000 V was reached. The electrode solu-
35 tions were 0.1 M NaOH for the cathode and 40 mM glutamic acid (pH 3.0) for the anode.
Transfer to nitrocellulose paper The IEF-gel was directly placed onto nitrocellulose paper and soaked into the electrophoretic chamber filled with electrode solution composed of 20 mM Tris, 150 mM glycine and 20% methanol [26]. The electrophoretic transfer was performed at 120 mA for 2 and 3 h when double and triple layers of nitrocellulose paper were used, respectively. The nitrocellulose paper was incubated for 24 h at 8°C, with 3% bovine serum albumin in TBS buffer containing 20 mM Tris-chloride, pH 7.5 + 0.5 M NaCI + 0.5% Triton + 1 mM CaCl 2 + 1 mM MgCI2 and I mM MnCI 2, and washed 3 × 15 rain with TBS buffer.
Detection of DT-diaphorase The isoforms of DT-diaphorase after discontinuous SDS-polyacylamide gel electrophoresis and IEF were detected by using protein-staining with Coomassie brillant blue R-250. The gel was directly fixed in a solution containing 5% sulphosalicylic acid+ 10% triehloroacetic acid for 60 rain and washed in the destaining solution, containing methanol: acetic acid: distilled water (3:1:6). The gel was stained in 0.001% Coomassie brillant blue R-250 dissolved in the destaining solution 24 h and destained until the background had completely disappeared. DT-diaphorase, transferred to nitrocellulose paper after IEF, was detected with three different methods: enzymatically with 344,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTF), by immunodetection and carbohydrate detection using lectins. The enzymatic staining with MTT was performed by incubating the nitrocellulose paper in a solution containing 50 mM Tris-chloride (pH 7.5), 2 mM MTF, 1 mM NADH and 3 p.M menadione. The reaction was stopped with 5% acetic acid. When immunodetection was performed, nitrocellulose paper was washed 3 x 15 rain with TBS buffer and incubated overnight with antibodies diluted in 20 mM Tris-chloride, pH 7.5 + 0.5 M NaCl + 0.1% Triton + 1 mM CaCl 2 + 1 mlVl MgC!z + 1% bovine serum albumin + 0.02% SDS and 1 mM MnCl 2. The nitrocellulose sheet was washed 3 x 15 min with TBS buffer and incubated 1 h with protein A labeled with 1~I diluted in 20 mM Tris-chloride, pH7.5 + 0.5M NaCl + 0 . 1 % T f i t o n + l mM CaC! 2 + 1 mM MgCl 2 + 1% bovine serum albumin + 0.02% SDS and 1 mM MnCI 2. The binding of antibodies to isoforms of DT-diaphorase was detected by ~ZSllabeled protein A using Raytest-TLC-scanner (lsotopenm-/~-ger~ite, Straubenhardy, Germany) equipped with radio-TLC-analyzer RITA-90 (Raytek Scientific Limited, Sheffield, U.K.) and autoradiography. The antibodies used in all the
experiments were raised against a homogenous DT-diaphorase purifi,:d from 1,,t liver (as revealed by SDSpolyacylamide gel electrophoresis and a ratio of absorbanee at 270 and 450 nm [15]) This enzyme preparation contained a mixture of the isoforms of DT-diaphorase. Carbohydrate detection was performed by incubating nitrocellulose paper with lectins diluted 1:200 in TBS buffer for 90 min. The samples were washed 3 x 15 min with the same buffer before and after incubation [60 min) with avidin-alkaline phosphatase, which was diluted l : 1000. The color development solution was prepared by mixing, just prior to use, l ml p-nitro blue tetrazolium chloride (NBT, 30 mg/ml) dissolved in 70% N,N-dimethylformamide (DMF) and 1 ml 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt (BCIP, 15 mg/ml) dissolved in DMF into 100 ml 0.1 M NaHCO 3, pH 9.8 + 1.0 mM MgCI 2. Two proteins from type A erythrocytes which bind to dolichos biflorus agglutinin (DBA) [27] were used as control and the specificity of this binding was determined using an inhibitor, 1 M N-acetylgalactosamine, specific for lectin DBA. Results
DT-diaphorase can be purified to apparent homogeneity from rat liver by a two-step purification procedure [15] where the key isolation step is the biospecific adsorption of the enzyme to immobilized dicoumarol, a competetive inhibitor of the enzyme with respect to NAD(P)H. In order to separate DT-diaphorase isoforms on the basis of differences in their net charges the eluate from the first purification step, i.e. an azodicoumarol-Sepharose 6B column, was applied to an FPLC-chromatofocusing column. Elution of this column with a pH gradient between pH 7.3 and 4.8 revealed 6 peaks (II to VII; Fig. 1). The first peak (Ia) was eluted with the void volume, while the second peak (Ib) was eluted directly after the void volume, indicating that the isoelectric points of these proteins were higher than pH 7.3. In fact, analysis of these peaks by isoelectric focusing technique showed isoelectric points of 9.1 and 7.5 for the protein in peak la and Ib, respectively. These proteins probably also have higher molecular weights than those in peak II-VII, since they can be removed by gel filtration on a Sephacryl S-200 column, the second step in the purification of DT-diaphorase [15], i.e., when the eluate from the Sephacryl S-200 column is applied to the FPLC-ehromatofocusing column neither peak la nor peak Ib can be detected in the eluate (not shown). Analysis of peak la by SDS-polyacrylamide gel dectrophorcsis revealed three bands with apparent molecular weights of 55 000, 43 000 and 30 000 (not shown). Although the proteins in peaks ia and Ib were eluted
36 VIb
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33
© 1992Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34138
Separation and characterization of isoforms of DT-diaphorase from rat liver cytosol Juan Segura-Aguilar a, Rudolf Kaiser b and Christina Lind a a Biochemical Toxicology, Wallenberg Laboratory, Unh'ersity o f Stockholm, Stockholm (Sweden) and h Department of Chemistry L Karolinska Institute, Stockholm (Sweden)
(Received 16 August 1991)
Keywords: Diaphorase;lsoform;Carbohydrate;DT-diaphorase;(Enzyme) Rats were treated with 3-methylcholanthrene {MC) and DT-diaphorase from liver was partially purified on an azodicoumarolSepharose 6B column and applied to an FPLC-chromatofocusing column in order to resolve isoforms. Six peaks showing significant DT-diaphorase activity were eluted from this column with a pH gradient between 7.30 to 4.80. The amino acid compositions of the two major peaks (II and Vlb) were found to be nearly identical, suggesting existence of isoforms rather than isozymes of DT-diaphorase. The isoforms of DT-diaphorase showed broad substrate specificities towards four different quinones (menadione, vitamin K-I, benzo(a)pyrene 3,6-quinone and cyclized-dopamine ortho-quinone), although quantitative differences in the specific activities were also found. All isoforms arc glycoproteins but contain different carbohydrates. Thus isoform II reacts with biotinylated lectins which are specific for N-acetylgalactosamine, mannose, fucose and galactosyl(fl-l,3)N-acetylgalactosamine, while isoform Vlb reacts only with biotinylated lectins specific for mannose and N-acetylgalaetosamine. Separation of DT-diaphorase isoforms from control rat liver cytosol using FPLC-ehromatofocusing revealed that the induction of the isoforms is not uniform, since isoform II was not found and the major isoform was composed of three peaks, whereas the major isoform of DT-diaphorase from liver cytosol of rats treated with 3-methylcholanthrene was composed of or~;: -.wo oeaks.
Introduction DT-diaphorase (NAD(P)H-quinone oxidoreductase, EC 1.6.99.2) is a flavoprotein which catalyzes the two-electron reduction of quinones, quinone epoxides and azo dyes [1-3]. This enzyme, which is induced by 3-methylcholanthrene and a large number of xenobiotics (see Refs. 4 and 5 for a review), seems to play a protective role against carcinogenicity [6], mutagenicity [7] and cytotoxicity [8] caused by quinones and their metabolic intermediates. The protective role of this enzyme seems to depend on its ability to reduce
Abbreviations: BP3,6-Q, benzo(a)pyrene 3,6-quinone; cDAoQ, cyclized doparaine ortho-quinone; DBA, dolichos biflorus agglutinin: Con A, cauavalia ensiformis agglutinin; FPLC, fast-protein liquid chromatography; IEF, isoelectric focusing; K3, raenadione; MC, 3raethylcholanthrene; MTT, 3-(4,5-diraethylthiazol-2-yl)-2,5-diphenyltetrazoliura bromide; PNA, peanut agglutinin; RCA'I, ricinus comraunis agglutininI; SBA, soybeanagglutinin;UEA 1, ulexeuropaeus agglutinin I; vit. K-l, vitamin K-I; WGA,wheat germ agglutinin. Correspondence: J. Segura-Aguilar,BiochemicalToxicology,Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm,Sweden.
quinones to hydroquinones, which are the substrates for conjugation reactions by UDP-glucuronosyl transferases and phenoisulfotranferases [9-11], and on its ability to compete with enzymes such as NADPH-cytochrome /-450 oxidoreductase, NADH-cytochrome b 5 oxidoreductase and NADH-ubiquinone oxidoreductase, which generate reactive species of oxygen when catalyzing the one-electron reduction of quinone to semiquinone [8,12]. Evidence supporting the existence of different forms of DT-diaphorase has appeared in several reports. In 1978 two different N-terminal amino acids of DT-diaphorase were reported [13]. In 1980 DT-diaphorase was reported to give rise to two distinct precipitates when antisera produced against rat liver cytosol was used in immunodifussion techniques [14]. In 1981 three different antigenic forms of isolated cytosolic as well as microsomal DT-diaphorase could be detected [15,16] whereas mitochondrial DT-diaphorase contained only two different antigenic forms [15]. In 1986 the separation of two isoforms of DT-diaphorase from murine liver cytosol, a hydrophilic and a hydrophobic form, was reported [17]. In 1987 the separation of several isoforms of DT-diaphorase on the basis of differences in their net charges was achieved [18]. The eDNA
sequences of one gene coding for DT-diaphorase [!921] from rat and human liver cytosol indicate that the various isoforms of DT-diaphorase reflect different posttranslational modifications rather than differences in primary structures. This study presents a luther characterization of DT~diaphorase isoforms and reports the separation of two additional dicoumarobbinding proteins by FPLCchromatofocusing. Materials and Methods
Chemicals Dopamine and 3-methylcholanthrene (MC) were obtained from Fluka AG (Buchs, Switzerland), Agarose C, Pharmalyte 3-10 and Polybuffer 74, low molecular weight calibration kit and isoelectric focusing calibration kit were from Pharmacia (Uppsala, Sweden). Menadione, NADH and vitamin K-l were purchased from Sigma (St Louis, MO, U.S.A.) and benzo(a)pyrene-3,6-quinone was from lIT Research Institute (Chicago, IL, U.S.A.). Lectins were from Vectors Laboratories Incorporated (Burlingame, U.S.A.) and avidin-alkaline phosphatase, 5-bromo-4-chloro-3indoyl phosphate p-toluidine salt (BCIP) and p-nitro blue tctrazolium chloride (NBT) were from Bio-Rad (California, U.S.A.). Endoglycosidase F and Endo-aN-acetylgalactosaminidase were obtained from Boehringer Mannheim (Mannheim, Germany).
Animals Male Sprague-Dawley rats of about 100 g body weight were used. The animals were kept on standard laboratory diet and starved for 24 h before death. Some animals were injected i,p. with 2 mg MC (dissolved in 0,25 m l corn oil)once daily for 3 days and decapitated 24 h after the last injection.
Isolation of DT-diaphorase isoforms by chromatofocusing Throughout this investigation liver cytosol from MC-treated and control rats has been used. Cytosol was prepared acording to Ernster et al. [22]. Cytosolic DT-diaphorase was purified as described earlier [15] by using adsorption to immobilised dicoumarol, a competitive inhibitor of the enzyme with respect to NAD(P)H. Elution of this azodicoumarol Sepharose 6B column with NADH resulted in an almost pure enzyme preparation and the recovery was about 80%. The eluate from the azodicoumarol-Sepharose 68 column was concentrated by ultrafiltration in a diaflo cell (Amicon, Lexington MA, U.S.A.) equipped with a PM-30 filter. The concentrated sample was applied to a Sephadex G-25 column equilibrated with 0.025 M imidazole-HCI, pH 7.3 + 0.25 M sucrose in order to remove NADH. This eluate was applied, at a rate of 60 m l / h, to a
mono P FP!_C chromatofocusing column (0.5 x 20 cm, Pharmacia, Sweden) equilibrated with 0.025 M imidazole-HCi, pH 7.3 + 0.25 M sucrose and eluted with polybuffer 74, pH 4.80 + 0.25 M sucrose, at 4°C.
DT-diaphorase acticity DT-diaphorase activity was measured essentially as described by Ernster et al. [1]. The standard assay system contained 50 mM Tris-HCI (pH 7.5), 0.08% Triton X-100, 500 # M NADH, 10 # M menadione and 77/zM cytochrome c. The reaction was measured spectrophotometrically by following the reduction of cytochrome c, which continuously reoxidizes menadione, at 550 nm and employing an extinction coefficient of 18.5 mM - i cm - J. When vitamin K-1 or benzo(a)pyrene 3,6-quinone was used as an electron acceptor, 0.020 mM was presented in the reaction mixture, where Triton X-100 was replaced by 0.25 M sucrose. When cDAoQ was used as an electron acceptor, DT-diaphorase activity was measured as recently reported [23], using 50 mM Tris-HCl, pH 6.5, + 0.25 M sucrose, 10 #M dopamine and 40 /~M Mn3+-pyrophosphate complex. After 2 min, 200 #M NADH was added to this reaction mixture and the reaction was started by addition of the enzyme. The reaction was followed at 475 nm and an extinction coefficient of 3058 M -~ cm- ~ was employed.
Analysis of amh~o acid composition Total amino acid composition of the isoforms of DT-diaphorase was determined with a Beckman 121M amino acid analyzer after hydrolysis for 24 h at 110°C in evacuated tubes with 6 M HCl/0.5% phenol, as described earlier [24].
Carbohydrate digestion by N- and O-glycosidases Isoforms of DT-diaphorase were incubated for 24 h at 37°C in phosphate bufter, pH 7.2 + 50 mM EDTA in the presence and in the absence of endoglycosidase F and analysed by isoelectric focusing (IEF, see below). When N-acetylgalactosaminidase was used, the isoforms were incubated in 20 mM phosphate buffer (pH 6.5) at 37°C for 24 h in the presence and in the absence of 0.1% SDS and 1% 2-mercaptoethanol. Two glycoproteins, ovalbumin and asialofetuin, were used as controls for endoglycosidase F and N-acetylgalactosaminidase activities, respectively.
Electrophoresis and isoelectric focusing (IEF) Discontinous SDS-polyacylamide gel electrophoresis was performed according Laemmli [25]. The polyacrylamide gel used for isoelectric focusing (IEF) was 1.5 mm thick and contained 5% acrylamide +0.15% bisacrylamide, 14% glycerol and 6.3% Pharmalyte 3-10. The gel was prefocused for 60 min at 7 W and focused at 7 W until 2000 V was reached. The electrode solu-
35 tions were 0.1 M NaOH for the cathode and 40 mM glutamic acid (pH 3.0) for the anode.
Transfer to nitrocellulose paper The IEF-gel was directly placed onto nitrocellulose paper and soaked into the electrophoretic chamber filled with electrode solution composed of 20 mM Tris, 150 mM glycine and 20% methanol [26]. The electrophoretic transfer was performed at 120 mA for 2 and 3 h when double and triple layers of nitrocellulose paper were used, respectively. The nitrocellulose paper was incubated for 24 h at 8°C, with 3% bovine serum albumin in TBS buffer containing 20 mM Tris-chloride, pH 7.5 + 0.5 M NaCI + 0.5% Triton + 1 mM CaCl 2 + 1 mM MgCI2 and I mM MnCI 2, and washed 3 × 15 rain with TBS buffer.
Detection of DT-diaphorase The isoforms of DT-diaphorase after discontinuous SDS-polyacylamide gel electrophoresis and IEF were detected by using protein-staining with Coomassie brillant blue R-250. The gel was directly fixed in a solution containing 5% sulphosalicylic acid+ 10% triehloroacetic acid for 60 rain and washed in the destaining solution, containing methanol: acetic acid: distilled water (3:1:6). The gel was stained in 0.001% Coomassie brillant blue R-250 dissolved in the destaining solution 24 h and destained until the background had completely disappeared. DT-diaphorase, transferred to nitrocellulose paper after IEF, was detected with three different methods: enzymatically with 344,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTF), by immunodetection and carbohydrate detection using lectins. The enzymatic staining with MTT was performed by incubating the nitrocellulose paper in a solution containing 50 mM Tris-chloride (pH 7.5), 2 mM MTF, 1 mM NADH and 3 p.M menadione. The reaction was stopped with 5% acetic acid. When immunodetection was performed, nitrocellulose paper was washed 3 x 15 rain with TBS buffer and incubated overnight with antibodies diluted in 20 mM Tris-chloride, pH 7.5 + 0.5 M NaCl + 0.1% Triton + 1 mM CaCl 2 + 1 mlVl MgC!z + 1% bovine serum albumin + 0.02% SDS and 1 mM MnCl 2. The nitrocellulose sheet was washed 3 x 15 min with TBS buffer and incubated 1 h with protein A labeled with 1~I diluted in 20 mM Tris-chloride, pH7.5 + 0.5M NaCl + 0 . 1 % T f i t o n + l mM CaC! 2 + 1 mM MgCl 2 + 1% bovine serum albumin + 0.02% SDS and 1 mM MnCI 2. The binding of antibodies to isoforms of DT-diaphorase was detected by ~ZSllabeled protein A using Raytest-TLC-scanner (lsotopenm-/~-ger~ite, Straubenhardy, Germany) equipped with radio-TLC-analyzer RITA-90 (Raytek Scientific Limited, Sheffield, U.K.) and autoradiography. The antibodies used in all the
experiments were raised against a homogenous DT-diaphorase purifi,:d from 1,,t liver (as revealed by SDSpolyacylamide gel electrophoresis and a ratio of absorbanee at 270 and 450 nm [15]) This enzyme preparation contained a mixture of the isoforms of DT-diaphorase. Carbohydrate detection was performed by incubating nitrocellulose paper with lectins diluted 1:200 in TBS buffer for 90 min. The samples were washed 3 x 15 min with the same buffer before and after incubation [60 min) with avidin-alkaline phosphatase, which was diluted l : 1000. The color development solution was prepared by mixing, just prior to use, l ml p-nitro blue tetrazolium chloride (NBT, 30 mg/ml) dissolved in 70% N,N-dimethylformamide (DMF) and 1 ml 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt (BCIP, 15 mg/ml) dissolved in DMF into 100 ml 0.1 M NaHCO 3, pH 9.8 + 1.0 mM MgCI 2. Two proteins from type A erythrocytes which bind to dolichos biflorus agglutinin (DBA) [27] were used as control and the specificity of this binding was determined using an inhibitor, 1 M N-acetylgalactosamine, specific for lectin DBA. Results
DT-diaphorase can be purified to apparent homogeneity from rat liver by a two-step purification procedure [15] where the key isolation step is the biospecific adsorption of the enzyme to immobilized dicoumarol, a competetive inhibitor of the enzyme with respect to NAD(P)H. In order to separate DT-diaphorase isoforms on the basis of differences in their net charges the eluate from the first purification step, i.e. an azodicoumarol-Sepharose 6B column, was applied to an FPLC-chromatofocusing column. Elution of this column with a pH gradient between pH 7.3 and 4.8 revealed 6 peaks (II to VII; Fig. 1). The first peak (Ia) was eluted with the void volume, while the second peak (Ib) was eluted directly after the void volume, indicating that the isoelectric points of these proteins were higher than pH 7.3. In fact, analysis of these peaks by isoelectric focusing technique showed isoelectric points of 9.1 and 7.5 for the protein in peak la and Ib, respectively. These proteins probably also have higher molecular weights than those in peak II-VII, since they can be removed by gel filtration on a Sephacryl S-200 column, the second step in the purification of DT-diaphorase [15], i.e., when the eluate from the Sephacryl S-200 column is applied to the FPLC-ehromatofocusing column neither peak la nor peak Ib can be detected in the eluate (not shown). Analysis of peak la by SDS-polyacrylamide gel dectrophorcsis revealed three bands with apparent molecular weights of 55 000, 43 000 and 30 000 (not shown). Although the proteins in peaks ia and Ib were eluted
36 VIb
04
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I
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l. .~. ,t , "~. ,
~
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I 20 0FRACTION NUMBER L
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