/. Biochem., 81, 169-177 (1977)
Purification, Crystallization, and Isolation of a Binary Complex with NADH Hiroyuki HASEGAWA1 Department of Biology, Tokyo Metropolitan University, Fukasawa, Setagaya-ku, Tokyo 158 Received for publication, June 10, 1976
Dihydropteridine reductase [EC 1.6 99.7] was purified from bovine liver in 50% yield and crystallized. The physicochemical properties of the purified enzyme were quite similar to those of sheep liver dihydropteridine reductase. During the course of purification, however, the enzyme was found to be separated into 2 major peaks together with minor peaks by column chromatography on CM-Sephadex, and one of the major peaks was identified as a binary complex of the enzyme with NADH. The reductase-NADH complex was also prepared in vitro and crystallized. Upon addition of quinonoid-dihydropterin to the complex, NADH was oxidized and released from the enzyme The amount of bound NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH complex in bovine liver extract as a predominant form was in accord with the pyridine nucleotide specificity for NADH as a coenzyme. The results further support the view that NADH is the natural coenzyme of this reductase.
It has been established by Kaufman and co-workers that tetrahydroptenn acts as an electron donor in the reaction catalyzed by phenylalanine hydroxylase [EC 1.14.16 1] in the liver. Quinonoid-dihydropterin, which is formed from tetrahydroptenn during phenylalanine hydroxylation, was shown to be reduced back to tetrahydroform by dihydropteridine reductase [EC 1.6.99.7] in the presence of NADPH or NADH (/). The reductase was purified from the extracts of mammalian liver, adrenal medulla 1
Present address' Department of Biochemistry, Hamamaisu University School of Medicine, 3600 Handa-cho, Hamamatsu, Shizuoka 431-31. Abbreviations: Ptenn, 2-amino-4-hydroxyptendine; 6MPH4, 5,6,7,8-tetrahydro-6-methylptenn; MTT-tetrazolium, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazoliumbromide; DCPIP, 2, 6-dichlorophenolindophenol. Vol. 81, No. 1, 1977
and brain, and the preparations from these organs showed common physicochemical properties and specificity for NADH as a coenzyme (2, 3). In the present study, however, the reductase was found to exist in multiple forms in crude enzyme preparations from the bovine liver extract. This paper describes an improved method for purifying the reductase from bovine liver. In addition, evidence is presented indicating that the predominant form of the enzyme in the extract is a binary complex with NADH. MATERIALS AND METHODS
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Dihydropteridine Reductase from Bovine Liver
Assay on Dihydropteridine Reductase—The activity was assayed by the tetrahydropterindepsndent reduction of fern-cytochrome c in the presence of NADH (4, 5). The standard assay 169
170
adequate for the present purpose because the reaction with tetrahydropterin was sluggish. 6-Methylpterin synthesized by the method of Seeger et al. (7) was reduced to the tetrahydro-form by catalytic hydrogenation (8). Quinonoid-dihydropterin was prepared by oxidation of 6MPH4 with 2,6-dichlorophenohndophenol (DCPIP) (9), and separated from excess DCPIP and the reduced dye by passage through a column of Sephadex G-25 equilibrated with cold 0.1 M Tris-HCl (pH 7.0). For the separation of 0.5-1 ml of pterin-DCPIP mixture, 4-5 ml of the gel was sufficient. Acrylamide Disc Gel Electrophoresis—Analytical acrylamide disc gel electrophoresis was carried out according to Davis (JO). A current of 2 milliamperes per tube was supplied at 2°. Proteia was stained with Amido-Black 10B. Dihydroptendine reductase was located on the gel by the deposition of formazan formed from M'l'ltetrazolium by reduction with tetrahydropterin as follows. After electrophoresis the gel was immersed in a cold solution containing MTT-tetrazolium (0.5 mg/ml), 0.5 min NADH, and 0.25 M Tris-HCl (pH 7.6). Freshly prepared quinonoiddihydropterin was added to give a final concentration of about 10 //M. The mixture was incubated at 30° for 10 min. The reaction was stopped byreplacing the medium with 7% acetic acid. The blue activity band that appeared on the gel was stable for 24 h in the dark. Sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDSelectrophoresis) was performed according to Weber and Osborn (77). Ovalbumin, trypsin [EC 3.4.21.4], lysozyme [EC 3.2.1.17], pepsin [EC 3.4.23.1], lactate dehydrogenase [EC 1.1.1.27], and catalase [EC 1.11.1.6] were used as marker proteins. Polypeptide chain molecular weights of the markers were taken from the table presented byWeber and Osborn (11).
Preparations—Fresh bovine livers were obtained from a slaughterhouse, immediately packed Molecular Weight Determination—Gel filtraon dry ice and stored at —20° until use. Reduced tion chromatography was carried out on a column cytochrome c, prepared from yeast by the method of Sephadex G-100 (1.5x90 cm) equilibrated with of Hagihara et al. (6), was a kind gift from Dr. H. 10 mM potassium phosphate containing 0.1 M KCT Mizushima and Mr. J. Tohtsu, Central Research (pH 6.5). The following proteins were used as Laboratories of Sankyo Co. The reduced cyto- standards (molecular weight in parentheses): bovine chrome c was oxidized with potassium ferricyanide scrum albumin (68,000), ovalbumin (44,000), pepsin and the reagent was removed by passage through a (34,000), trypsin (24,000), lysozyme (14,300), and .Sephadex G-25 column equilibrated with 0.5 M KC1. cytochrome c (11,700). Among the cytochrome c preparations tested, the Equilibrium sedimentation was carried out "acid modified" one (Sigma, Type XII) was in- with a Beckman model E analytical ultracentnfuge. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/169/835887 by Stockholm University Library user on 19 January 2019
mixture (2 ml) contained 80 //moles of Tris-HCl (pH 7.6), 0.1 //moles of ferri-cytochrome c, 0 1 //moles of NADH, 2 nmoles of tetrahydro-6methylpterin (6MPH4), and the enzyme. When the activity of the purified enzyme preparation was measured, the components other than the enzyme were put into a cuvette in the order mentioned above and the mixture was left to stand for 30 sec to ensure oxidation of 6MPH4 to the corresponding quinonoid-dihydropterin by cytochrome c. The reaction was then initiated by the addition of enzyme, and the increase of absorbance at 550 run was followed at 25° (assay system I). When the crude liver extract was used as enzyme, NADH- and 6MPH4independent reduction of cytochrome c occurred and persisted for a few minutes. The reductase reaction was, therefore, initiated by the addition of 6MPH4 after the extract had been incubated for 3-4 min With the other components (assay system II). In each case, the reduction of cytochrome c proceeded linearly with time, after a lag period of several seconds (assay system I) or after an initial burst of reduction (assay system IT). The enzyme activity was calculated from the linear region. The steady-state velocity of cytochrome c reduction was a linear function of the amount of enzyme up to an activity of 6 nmoles of cytochrome c reduced per minute under the standard assay conditions. In all experiments, control incubations were carried out to correct for both 6NfPH4-independent reduction of cytochrome c and reduction due to the spontaneous formation of tetrahydropterin from quinonoid-dihydropterin with NADH. Enzyme preparations were diluted with cold 50 mM Tris-HCl (pH 7.6) to give an activity of 1-5 nmoles cytochrome c reduced per minute per 10 //I of the enzyme, left to stand for 2 min in the cold and then used for the assay.
H. HASEGAWA.
DIHYDROPTERIDINE REDUCTASE
2-5°. Centrifugation was performed with a Hitachi 18-PRrefrigeratedcentrifuge at 7,700 x g. Dialysis was performed overnight with two changes of the medium, unless otherwise stated. Extraction: Partially thawed bovine liver (1 kg) was cut into small pieces and 250 g was homogenized with 350 ml of cold 20 mM acetic acid for 30 sec in a Waring blender (model 5011). The blending was continued for a further 30 sec after the addition of 400 ml of the acetic acid solution. In order to avoid foaming, the mixture was covered with a sheet of polyethylene film during blending. The homogenate was centrifuged for 60 nun and the pH of the supernatant solution was adjusted to 7.4 with 1 M Tris. Chloroform-ethanol fractwnation: A mixture of 210 ml of ethanol (—70°) and 130 ml of chloroform (—20°) was added per liter of the crude extract. The mixture was vigorously shaken for 1 min and then centrifuged for 15 min. The combined aqueous layer was dialyzed against 40 liters of distilled water for 3 h and then against 40 liters of 10 mM potassium phosphate (pH 6.8) overnight witu a change of the buffer. Solid ammonium sulfate was added to the dialyzed solution to 80 % saturation. After stirring for 30 min, the precipitate was collected by centrifugation for 50 min, transfered to a Visking tube and dialyzed against 10 mM potassium phosphate (pH 6.8). The precipitate began to dissolve soon after the start of dialysis. Ammonium sulfate fractionation: The dialyzed solution was diluted with 10 mM potassium phosphate (pH 6.8) to give a protein concentration of about 30 mg/ml. Solid ammonium sulfate was added (34 g per 100 ml) and after stirring for 1 h, the precipitate was removed by centrifugation for 40 min. Additional ammonium sulfate was added to the supernatant until most of the enzyme had precipitated (usually 8 g per 100 ml of the original solution). The precipitate was collected and dialyzed extensively against 40 mM potassium phosphate (pH 7.4) as described above. DEAE-Sephadex A-50 column chromatography: The dialyzed solution (79 ml) was applied to a DEAE-Sephadex A-50 column (2.5 x 40 cm) equilibrated with 40 mM potassium phosphate (pH 7.4). RESULTS Following application of the enzyme, the column Purification of Bovine Liver Dihydropteridine was washed with 250 ml of equilibrating buffer. A Reductase—All manipulations were carried out at 750-ml concave gradient from 0.0 to 0.25 M KC1 in
Vol. 81, No. 1, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/169/835887 by Stockholm University Library user on 19 January 2019
Centrifugation was performed at 8,225 rpm at 15°. Before centrifugation, the enzyme was dialyzed against 5 mM Tris-HG containing 0.1 M KC1 (pH 7.6). The partial specific volume of the enzyme (0.73 ml/g) was calculated from the ammo acid composition. Amino Acid Analysis—The purified enzyme was hydrolyzed with 6 N HC1 at 105° for 21-22 h and the hydrolyzate was subjected to amino acid analysis using a JEOL 6AH automatic amino acid analyzer. The amounts of serine, threonine, and tyrosine were corrected based on the data of Hirs et •al. (12). Tryptophan was determined spectrophotometrically according to Goodwin and Morton (13). Miscellaneous Analytical Methods—The amount of purified dihydropteridine reductase was •determined from the extinction coefficient (see beJow). For other preparations, protein was estijnated by the original method of Warburg and •Christian (14). NAD + was determined using the •ethanol-alcohol dehydrogenase system at pH 8.5. Tetrahydropterin was determined by spectrophotojnetric titration with DCPIP (15). The following molar extinction coefficients were used: NADH, -6,200 at 340 nm; DCPIP, 19,500 at 602 nm (pH 7.6); cytochrome c, 21,000 (reduced—oxidized) at .550 nm (16). Reagents—NADH, NADPH (Oriental Yeast •Co.), MTT-tetrazohum (Dohjin Laboratories), DCPIP (Wako Pure Chemical Industries), alcohol •dehydrogenase [EC 1.1.1.1, from yeast, Boehringer Mannheim], and glucose-6-phosphate dehydrogeJiase [EC 1.1.1.49, from yeast, Oriental Yeast Co.] •were purchased from the sources shown. Marker proteins for molecular weight determination were •obtained from the following sources: Serum .albumin (bovine, cryst.) and ovalbumin (egg white, -2xcryst.) from Calbiochem; lysozyme (egg white, 2 x cryst.) from Tokyo Kasei Kogyo; trypsin from Worthington; lactate dehydrogenase (bovine heart) from Worthington; catalase (bovine liver, 2x •cryst.) was a gift from Dr. T. Ohoka of Tokyo Metropolitan University. Hydroxylapatite was prepared by the method of Tizelius et al. (17).
171
H. HASEGAWA
172
TABLE I.
Physicochemical Properties of Bovine Liver Dihydropteridine Reductase—Gel electrophoresis:
The purified reductase migrated as a single protein in an electric field and the activity band corresponded exactly to that of protein. The protein gave a single band upon SDS-electrophoresis; the molecular weight of the subunit was estimated tobe 25,000 based on its mobility relative to those of marker proteins (//). No visible bands appeared on the gel on staining with the periodic acid-Schiff
Purification of dihydropteridine reductase from bovine liver.
Step of purification ].
with 50mM Tns-HCl (pH 7 4). Following theapplication of the enzyme, the column was washed with 100 ml of the equilibrating buffer and then the enzyme was eluted with a 300-ml linear gradient from 0.0 to 0.3 M KC1 in 50 mM Tns-HCl (pH 7.4> at a flow rate of about 10 ml/h (5-ml fractions). Active fractions (115-150 ml of eluate) were combined, concentrated to 4-5 ml and dialyzed against 50 mM Tris-HCl (pH 7.4). Typical data on the purification of bovine liver dihydropteridine reductase are summarized in Table I. The purified enzyme was stable at -70° for at least 2 years. Physicochemical properties were examined using the final preparation, unless otherwise stated. In the assay procedure for the enzyme, 6MPH4 was quantitatively replaceable by the corresponding quinonoid-dihydroptenn. 7,8-Dihydroptennstested, including dihydrofolate and dihydrobioptenn, were inactive. The ptenn-independent activity observed in the crude enzyme preparations(10% in the crude extract) was removed by the purification procedures and was not detectable in the DEAE-Sephadex fraction.
Volume (ml)
Total protein (mg)
Total activity (units")
Specific activity (units
Purification, Crystallization, and Isolation of a Binary Complex with NADH Hiroyuki HASEGAWA1 Department of Biology, Tokyo Metropolitan University, Fukasawa, Setagaya-ku, Tokyo 158 Received for publication, June 10, 1976
Dihydropteridine reductase [EC 1.6 99.7] was purified from bovine liver in 50% yield and crystallized. The physicochemical properties of the purified enzyme were quite similar to those of sheep liver dihydropteridine reductase. During the course of purification, however, the enzyme was found to be separated into 2 major peaks together with minor peaks by column chromatography on CM-Sephadex, and one of the major peaks was identified as a binary complex of the enzyme with NADH. The reductase-NADH complex was also prepared in vitro and crystallized. Upon addition of quinonoid-dihydropterin to the complex, NADH was oxidized and released from the enzyme The amount of bound NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH complex in bovine liver extract as a predominant form was in accord with the pyridine nucleotide specificity for NADH as a coenzyme. The results further support the view that NADH is the natural coenzyme of this reductase.
It has been established by Kaufman and co-workers that tetrahydroptenn acts as an electron donor in the reaction catalyzed by phenylalanine hydroxylase [EC 1.14.16 1] in the liver. Quinonoid-dihydropterin, which is formed from tetrahydroptenn during phenylalanine hydroxylation, was shown to be reduced back to tetrahydroform by dihydropteridine reductase [EC 1.6.99.7] in the presence of NADPH or NADH (/). The reductase was purified from the extracts of mammalian liver, adrenal medulla 1
Present address' Department of Biochemistry, Hamamaisu University School of Medicine, 3600 Handa-cho, Hamamatsu, Shizuoka 431-31. Abbreviations: Ptenn, 2-amino-4-hydroxyptendine; 6MPH4, 5,6,7,8-tetrahydro-6-methylptenn; MTT-tetrazolium, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazoliumbromide; DCPIP, 2, 6-dichlorophenolindophenol. Vol. 81, No. 1, 1977
and brain, and the preparations from these organs showed common physicochemical properties and specificity for NADH as a coenzyme (2, 3). In the present study, however, the reductase was found to exist in multiple forms in crude enzyme preparations from the bovine liver extract. This paper describes an improved method for purifying the reductase from bovine liver. In addition, evidence is presented indicating that the predominant form of the enzyme in the extract is a binary complex with NADH. MATERIALS AND METHODS
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/169/835887 by Stockholm University Library user on 19 January 2019
Dihydropteridine Reductase from Bovine Liver
Assay on Dihydropteridine Reductase—The activity was assayed by the tetrahydropterindepsndent reduction of fern-cytochrome c in the presence of NADH (4, 5). The standard assay 169
170
adequate for the present purpose because the reaction with tetrahydropterin was sluggish. 6-Methylpterin synthesized by the method of Seeger et al. (7) was reduced to the tetrahydro-form by catalytic hydrogenation (8). Quinonoid-dihydropterin was prepared by oxidation of 6MPH4 with 2,6-dichlorophenohndophenol (DCPIP) (9), and separated from excess DCPIP and the reduced dye by passage through a column of Sephadex G-25 equilibrated with cold 0.1 M Tris-HCl (pH 7.0). For the separation of 0.5-1 ml of pterin-DCPIP mixture, 4-5 ml of the gel was sufficient. Acrylamide Disc Gel Electrophoresis—Analytical acrylamide disc gel electrophoresis was carried out according to Davis (JO). A current of 2 milliamperes per tube was supplied at 2°. Proteia was stained with Amido-Black 10B. Dihydroptendine reductase was located on the gel by the deposition of formazan formed from M'l'ltetrazolium by reduction with tetrahydropterin as follows. After electrophoresis the gel was immersed in a cold solution containing MTT-tetrazolium (0.5 mg/ml), 0.5 min NADH, and 0.25 M Tris-HCl (pH 7.6). Freshly prepared quinonoiddihydropterin was added to give a final concentration of about 10 //M. The mixture was incubated at 30° for 10 min. The reaction was stopped byreplacing the medium with 7% acetic acid. The blue activity band that appeared on the gel was stable for 24 h in the dark. Sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDSelectrophoresis) was performed according to Weber and Osborn (77). Ovalbumin, trypsin [EC 3.4.21.4], lysozyme [EC 3.2.1.17], pepsin [EC 3.4.23.1], lactate dehydrogenase [EC 1.1.1.27], and catalase [EC 1.11.1.6] were used as marker proteins. Polypeptide chain molecular weights of the markers were taken from the table presented byWeber and Osborn (11).
Preparations—Fresh bovine livers were obtained from a slaughterhouse, immediately packed Molecular Weight Determination—Gel filtraon dry ice and stored at —20° until use. Reduced tion chromatography was carried out on a column cytochrome c, prepared from yeast by the method of Sephadex G-100 (1.5x90 cm) equilibrated with of Hagihara et al. (6), was a kind gift from Dr. H. 10 mM potassium phosphate containing 0.1 M KCT Mizushima and Mr. J. Tohtsu, Central Research (pH 6.5). The following proteins were used as Laboratories of Sankyo Co. The reduced cyto- standards (molecular weight in parentheses): bovine chrome c was oxidized with potassium ferricyanide scrum albumin (68,000), ovalbumin (44,000), pepsin and the reagent was removed by passage through a (34,000), trypsin (24,000), lysozyme (14,300), and .Sephadex G-25 column equilibrated with 0.5 M KC1. cytochrome c (11,700). Among the cytochrome c preparations tested, the Equilibrium sedimentation was carried out "acid modified" one (Sigma, Type XII) was in- with a Beckman model E analytical ultracentnfuge. J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/169/835887 by Stockholm University Library user on 19 January 2019
mixture (2 ml) contained 80 //moles of Tris-HCl (pH 7.6), 0.1 //moles of ferri-cytochrome c, 0 1 //moles of NADH, 2 nmoles of tetrahydro-6methylpterin (6MPH4), and the enzyme. When the activity of the purified enzyme preparation was measured, the components other than the enzyme were put into a cuvette in the order mentioned above and the mixture was left to stand for 30 sec to ensure oxidation of 6MPH4 to the corresponding quinonoid-dihydropterin by cytochrome c. The reaction was then initiated by the addition of enzyme, and the increase of absorbance at 550 run was followed at 25° (assay system I). When the crude liver extract was used as enzyme, NADH- and 6MPH4independent reduction of cytochrome c occurred and persisted for a few minutes. The reductase reaction was, therefore, initiated by the addition of 6MPH4 after the extract had been incubated for 3-4 min With the other components (assay system II). In each case, the reduction of cytochrome c proceeded linearly with time, after a lag period of several seconds (assay system I) or after an initial burst of reduction (assay system IT). The enzyme activity was calculated from the linear region. The steady-state velocity of cytochrome c reduction was a linear function of the amount of enzyme up to an activity of 6 nmoles of cytochrome c reduced per minute under the standard assay conditions. In all experiments, control incubations were carried out to correct for both 6NfPH4-independent reduction of cytochrome c and reduction due to the spontaneous formation of tetrahydropterin from quinonoid-dihydropterin with NADH. Enzyme preparations were diluted with cold 50 mM Tris-HCl (pH 7.6) to give an activity of 1-5 nmoles cytochrome c reduced per minute per 10 //I of the enzyme, left to stand for 2 min in the cold and then used for the assay.
H. HASEGAWA.
DIHYDROPTERIDINE REDUCTASE
2-5°. Centrifugation was performed with a Hitachi 18-PRrefrigeratedcentrifuge at 7,700 x g. Dialysis was performed overnight with two changes of the medium, unless otherwise stated. Extraction: Partially thawed bovine liver (1 kg) was cut into small pieces and 250 g was homogenized with 350 ml of cold 20 mM acetic acid for 30 sec in a Waring blender (model 5011). The blending was continued for a further 30 sec after the addition of 400 ml of the acetic acid solution. In order to avoid foaming, the mixture was covered with a sheet of polyethylene film during blending. The homogenate was centrifuged for 60 nun and the pH of the supernatant solution was adjusted to 7.4 with 1 M Tris. Chloroform-ethanol fractwnation: A mixture of 210 ml of ethanol (—70°) and 130 ml of chloroform (—20°) was added per liter of the crude extract. The mixture was vigorously shaken for 1 min and then centrifuged for 15 min. The combined aqueous layer was dialyzed against 40 liters of distilled water for 3 h and then against 40 liters of 10 mM potassium phosphate (pH 6.8) overnight witu a change of the buffer. Solid ammonium sulfate was added to the dialyzed solution to 80 % saturation. After stirring for 30 min, the precipitate was collected by centrifugation for 50 min, transfered to a Visking tube and dialyzed against 10 mM potassium phosphate (pH 6.8). The precipitate began to dissolve soon after the start of dialysis. Ammonium sulfate fractionation: The dialyzed solution was diluted with 10 mM potassium phosphate (pH 6.8) to give a protein concentration of about 30 mg/ml. Solid ammonium sulfate was added (34 g per 100 ml) and after stirring for 1 h, the precipitate was removed by centrifugation for 40 min. Additional ammonium sulfate was added to the supernatant until most of the enzyme had precipitated (usually 8 g per 100 ml of the original solution). The precipitate was collected and dialyzed extensively against 40 mM potassium phosphate (pH 7.4) as described above. DEAE-Sephadex A-50 column chromatography: The dialyzed solution (79 ml) was applied to a DEAE-Sephadex A-50 column (2.5 x 40 cm) equilibrated with 40 mM potassium phosphate (pH 7.4). RESULTS Following application of the enzyme, the column Purification of Bovine Liver Dihydropteridine was washed with 250 ml of equilibrating buffer. A Reductase—All manipulations were carried out at 750-ml concave gradient from 0.0 to 0.25 M KC1 in
Vol. 81, No. 1, 1977
Downloaded from https://academic.oup.com/jb/article-abstract/81/1/169/835887 by Stockholm University Library user on 19 January 2019
Centrifugation was performed at 8,225 rpm at 15°. Before centrifugation, the enzyme was dialyzed against 5 mM Tris-HG containing 0.1 M KC1 (pH 7.6). The partial specific volume of the enzyme (0.73 ml/g) was calculated from the ammo acid composition. Amino Acid Analysis—The purified enzyme was hydrolyzed with 6 N HC1 at 105° for 21-22 h and the hydrolyzate was subjected to amino acid analysis using a JEOL 6AH automatic amino acid analyzer. The amounts of serine, threonine, and tyrosine were corrected based on the data of Hirs et •al. (12). Tryptophan was determined spectrophotometrically according to Goodwin and Morton (13). Miscellaneous Analytical Methods—The amount of purified dihydropteridine reductase was •determined from the extinction coefficient (see beJow). For other preparations, protein was estijnated by the original method of Warburg and •Christian (14). NAD + was determined using the •ethanol-alcohol dehydrogenase system at pH 8.5. Tetrahydropterin was determined by spectrophotojnetric titration with DCPIP (15). The following molar extinction coefficients were used: NADH, -6,200 at 340 nm; DCPIP, 19,500 at 602 nm (pH 7.6); cytochrome c, 21,000 (reduced—oxidized) at .550 nm (16). Reagents—NADH, NADPH (Oriental Yeast •Co.), MTT-tetrazohum (Dohjin Laboratories), DCPIP (Wako Pure Chemical Industries), alcohol •dehydrogenase [EC 1.1.1.1, from yeast, Boehringer Mannheim], and glucose-6-phosphate dehydrogeJiase [EC 1.1.1.49, from yeast, Oriental Yeast Co.] •were purchased from the sources shown. Marker proteins for molecular weight determination were •obtained from the following sources: Serum .albumin (bovine, cryst.) and ovalbumin (egg white, -2xcryst.) from Calbiochem; lysozyme (egg white, 2 x cryst.) from Tokyo Kasei Kogyo; trypsin from Worthington; lactate dehydrogenase (bovine heart) from Worthington; catalase (bovine liver, 2x •cryst.) was a gift from Dr. T. Ohoka of Tokyo Metropolitan University. Hydroxylapatite was prepared by the method of Tizelius et al. (17).
171
H. HASEGAWA
172
TABLE I.
Physicochemical Properties of Bovine Liver Dihydropteridine Reductase—Gel electrophoresis:
The purified reductase migrated as a single protein in an electric field and the activity band corresponded exactly to that of protein. The protein gave a single band upon SDS-electrophoresis; the molecular weight of the subunit was estimated tobe 25,000 based on its mobility relative to those of marker proteins (//). No visible bands appeared on the gel on staining with the periodic acid-Schiff
Purification of dihydropteridine reductase from bovine liver.
Step of purification ].
with 50mM Tns-HCl (pH 7 4). Following theapplication of the enzyme, the column was washed with 100 ml of the equilibrating buffer and then the enzyme was eluted with a 300-ml linear gradient from 0.0 to 0.3 M KC1 in 50 mM Tns-HCl (pH 7.4> at a flow rate of about 10 ml/h (5-ml fractions). Active fractions (115-150 ml of eluate) were combined, concentrated to 4-5 ml and dialyzed against 50 mM Tris-HCl (pH 7.4). Typical data on the purification of bovine liver dihydropteridine reductase are summarized in Table I. The purified enzyme was stable at -70° for at least 2 years. Physicochemical properties were examined using the final preparation, unless otherwise stated. In the assay procedure for the enzyme, 6MPH4 was quantitatively replaceable by the corresponding quinonoid-dihydroptenn. 7,8-Dihydroptennstested, including dihydrofolate and dihydrobioptenn, were inactive. The ptenn-independent activity observed in the crude enzyme preparations(10% in the crude extract) was removed by the purification procedures and was not detectable in the DEAE-Sephadex fraction.
Volume (ml)
Total protein (mg)
Total activity (units")
Specific activity (units
