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[17] Dihydropteridine reductase from sheep brain
[17]
DIHYDROPTERIDINE REDUCTASE FROM SHEEP BRAIN
127
[17] D i h y d r o p t e r i d i n e R e d u c t a s e f r o m S h e e p B r a i n
By K. G. SCRIMGEOURand S. CHEEMA-DHADLI Introduction Dihydropterin reductase catalyzes the reduction of the unstable quinonoid dihydropterin formed during pterin-requiring hydroxylation reactions. Because NADH is the preferred reductant, the systematic name for the enzyme is NADH:6,7-dihydropteridine oxidoreductase (EC 1.6.99.7); however, it usually is called either dihydropterin or dihydropteridine reductase. Because tyrosine hydroxylase L2 and tryptophan hydroxylase 3-5 activities had been detected in brain tissue, we tested sheep brain as a potential source of the supplementary enzyme dihydropterin reductase, required for aromatic hydroxylations. The reductase was purified to homogeneity using a nine-step purification procedure, and the purified enzyme was compared to the dihydropterin reductase from sheep liver. 6 Quinonoid dihydropterin + N A D H + H + ~ 5,6,7,8-tetrahydropterin + N A D +
Assay Method
Principle Dihydropterin reductase activity was routinely measured by following the decrease in absorbance at 340 nm corresponding to oxidation of NADH. Quinonoid dihydropterin was prepared in situ in the reaction mixture from 6,7-dimethyltetrahydropterin, by oxidation with a stoichiometric amount of dichlorophenolindophenol. The quinonoid dihydropterin is then reduced back to the tetrahydropterin by NADH (chemical reaction) and by NADH in the presence of the reductase (enzymatic reaction). i T. N a g a t s u , M. Levitt, and S. Udenfriend, Biochem. Biophys. Res. Commun. 14, 543 (1964). 2 S. F a h n , J. S. R o d m a n , and L. J. Cote, J. Neurochem. 16, 1293 (1969). 3 D. G. G r a h a m e - S m i t h , Biochem. J. 105, 351 (1967). 4 D. A. V. Peters, P. L. McGeer, and E. C. McGeer, J. Neurochem. 15, 1431 (1968). 5 A. I c h i y a m a , S. N a k a m u r a , Y. N i s h i z u m a , and O. Hayaishi, J. Biol. Chem. 245, 1699 (1970). 6 S. C h e e m a , S. J. Soldin, A. K n a p p , T. H o f m a n n , and K. G. Scrimgeour, Can. J. Biochem. 51, 1229 (1973).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
128
CATABOLISM OF THE AROMATIC AMINO ACIDS
[17]
Reagents 6,7-Dimethyl-5,6,7,8-tetrahydropterin hydrochloride, 1.5 mM Dichlorophenolindophenol, 1.5 mM Tris-HC1, pH 6.8, 0.1 M NADH, 2 mM All reagents listed above were kept deaerated by bubbling with moist nitrogen gas. Procedure The reaction mixture contained 0.9 ml of Tris buffer, 0.05 ml of 6,7dimethyltetrahydropterin, 0.05 ml of NADH, 0.05 ml of dichlorophenolindophenol, and enough enzyme (e.g., 0.005 ml) to give a change in absorbance at 340 nm of 0.1 to 0.2/min. The enzyme was omitted from the reference cell. The reaction was initiated by the addition of the indophenol to both the experimental and reference cuvettes. The change in absorbance was recorded using a recording spectrophotometer with a scale expander and a constant temperature cell holder maintained at 25° using a thermostatted water bath. The reference cell contained all the components except the enzyme, and so provided the correction for the chemical reaction. For kinetic measurements, the initial velocity of each reaction was measured over an initial 10 to 20 sec, and a unit of activity was taken as that amount of enzyme that gave a change in absorbance of 0.62/ml/min due to the oxidation of NADH to NAD ÷. This is equivalent to the oxidation of approximately 0.1 /xmol of NADH. All assays were done in at least duplicate. Under the conditions of our assay, the oxidation of NADH by dichlorophenolindophenol was insignificant, so it was not necessary to apply the elaborate correction calculations used by Nielsen et al. 7 The reaction rates were linear over the time period used, and the initial velocities were directly proportional to the concentration of enzyme added. Purification Where possible, all manipulations were performed at 0-4 °. Frozen sheep brains (700 g) were partially thawed, and chopped into small pieces. The pieces were homogenized for 60 sec with 2 liters of cold 0.25 M sucrose in a Waring blender at one-third the full speed of the blender. The homogenate was centrifuged at 5000 rpm in a Lourdes Betafuge (4-liter head) for 20 min. The residue was mixed with another 1.5 liters of su7 K. H. Nielsen, V. S i m o n s e n , and K. E. Lind, Eur. J. Biochem. 9, 497 (1969).
[17]
D I H Y D R O P T E R I D I N E R E D U C T A S E F R O M S H E E P BRAIN
129
crose solution and homogenized again for another 20 sec. The second homogenate was then centrifuged and the two supernatant solutions were pooled. Solid ammonium sulfate was slowly stirred into the solution from the previous step until the final concentration of ammonium sulfate was 25%. This suspension was centrifuged and the residue was discarded. More ammonium sulfate was added to the supernatant fraction until 70% saturation was reached. The precipitate was collected by centrifugation and dissolved in about one-seventh the original volume of 0.02 M Tris buffer, pH 7.4 containing 10-3 M 2-mercaptoethanol, and dialyzed overnight against a large volume of the same buffer. [Mercaptoethanol (10 -3 M) was included in all buffers used in the purification.] Drops of zinc acetate (0.2 M) were stirred into the dialyzed solution until the ratio of zinc acetate solution to enzyme solution was 1 : 10. The suspension was stirred for 30 min until there was a thick cloudy precipitate, which was removed by centrifugation. The supernatant fraction was dialyzed overnight against two changes of 0.05 M Tris buffer, pH 7.4. To this dialyzed solution, solid ammonium sulfate (10 g/100 ml of solution) was added, and the pH of the solution was adjusted to 8.2 with 1 N ammonium hydroxide. Another 7 g of solid ammonium sulfate/100 ml of solution was added slowly. The precipitate was removed by centrifugation and discarded. After another 30 g of ammonium sulfate/100 ml of solution had been added, the solution was again centrifuged and the precipitate was dissolved in one-third the original volume of 0.01 M Tris buffer. To this solution, 0.2 M zinc acetate (4 ml/100 ml of protein solution) was added. A cloudy precipitate was seen after a few minutes and the solution was centrifuged. The supernatant fraction was dialyzed against 15 times its volume of 0.01 M Tris buffer, pH 7.4. Calcium phosphate gel (52.5 mg solid/ml) was added dropwise in the ratio of 20 ml of gel to 100 ml of the previous solution. The mixture was stirred for 30 min, centrifuged, and the supernatant fraction discarded. The gel was washed with 0.008 M phosphate, pH 6.8, and the supernatant solution again discarded. The gel was further washed with 0.1 M phosphate, pH 6.8, using a volume two-thirds that of the untreated enzyme solution. The supernatant solution obtained by centrifugation was dialyzed against a large volume of 0.03 M Tris buffer, pH 7.5. A column (2.5 x 40 cm) was packed with DEAE-Sephadex A-50 equilibrated with 0.03 M Tris buffer, pH 7.5. A dialyzed solution of enzyme (180 ml, 0.98 mg/ml) was applied to the column and eluted with a gradient of 500 ml of 0.05 M Tris buffer, in the mixing flask, and 500 ml of 0.3 M Tris, pH 7.5 in the reservoir. The enzyme was eluted at about 0.2 M buffer concentration with about a 10-fold purification.
130
CATABOLISM OF THE AROMATIC AMINO ACIDS
[17]
The fractions from the DEAE-Sephadex column containing enzyme activity were pooled and concentrated about 10-fold by ultrafiltration in a Diaflo apparatus. A column (1.5 x 90 cm) was packed with Sephadex G200 and equilibrated with 0.05 M Tris buffer, pH 7.5. The enzyme sample was applied in and eluted with 0.05 M Tris at 6 ml/hr. Of the two major protein peaks eluted, the second peak contained the enzyme activity. Those fractions containing activity were pooled and again concentrated in the Diaflo. Finally, the enzyme was purified by preparative polyacrylamide electrophoresis. The gel (5% acrylamide, 0.13% bisacrylamide, 0.007% fresh ammonium persulfate) was cast in a Pyrex glass tube (1.5 x 15 cm) open at both ends. After it was polymerized at room temperature, a 1 cm stacking gel was polymerized at the top of the column. 8 Ice-cold 0.03 M Tris-glycine buffer, pH 8.3, containing 10 -3 M 2-mercaptoethanol was used in both reservoirs and electrophoresis was performed at 0 to 4 ° at a constant current of 5 mA. Before application of the enzyme sample, electrophoresis of 0.5 ml of 0.002% bromphenol blue, which contained two or three drops of 40% sucrose, was carried out for 1 hr. This was to avoid mixing the enzyme sample with the tracking dye and also to get rid of the ammonium persulfate from the gel. 2-Mercaptoethanol (5/xl) and two or three drops of 40% sucrose were added to the enzyme sample before its application. The electrophoresis was carried out for 8 hr. During the electrophoresis, 5-/xl samples of 2-mercaptoethanol were added two or three times to the top buffer reservoir, to maintain the activity of the enzyme. At the end of the electrophoretic run, the gel was carefully removed from the tube and sliced into 1-2 mm slices. The slices were broken up by suspension in 2 ml of 0.05 M Tris buffer, pH 7.3, overnight at 4 °. The protein concentration of the supernatant solution was determined from its absorbance at 280 nm, and the enzyme activity was determined on 2-/zl aliquots. The gel slices containing the enzyme activity were extracted once or twice more. Usually only two slices contained most of the activity. The supernatant solutions from these were pooled and concentrated. The results of the purification procedure are summarized in Table I. The specific activity of a typical preparation was 61 units/mg, with an overall yield of about 5%. The sheep brain reductase was shown to be pure by analytical disc gel electrophoresis. The one band of protein coincided with activity of the enzyme, when activity was determined on slices of unstained gel. 8 B. J. Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).
[17]
DIHYDROPTERIDINE REDUCTASE FROM SHEEP BRAIN
131
TABLE 1 SUMMARY OF THE PURIFICATION OF qDHP REDUCTASE FROM SHEEP BRAIN"
Specific activity Step 1. Extract 2. Ammonium
Total volume (ml)
Total
Total
activity
protein
Yield
(units)
(mg)
(%)
0.9 !.5
3200 860
35,200 20,600
38,400 13,700
100 60
16.6 28.9
2.8 3.6
880 350
14,600 10,100
5,190 2,800
42 30
5.4
28
5.2
360
10,000
1,940
30
2.6
35.2
14
175
6,160
0.26 0.4 0.08
37.8 123 49.2
145 307 610
118 25 35
4,450 3,080 1,724
Protein
Number of
(mg/ml)
units/ml
12.6 16.4
11 24.1
5.9 8.0
(unit/mg protein)
sulfate 3. First zinc acetate 4. Alkaline ammonium sulfate 5. Second zinc
acetate 6. Calcium phosphate eluate 0.1 M
455
14.7
phosphate 7. DEAE-Sephadex 8. Sephadex G-200 9. Preparative disc
30.6 10 2.8
electrophoresis " Frozen sheep brain (700g).
Dihydropteridine reductase from beef kidney has been purified using immobilized Cibacron Blue. 9 Because of the similarities of brain and sheep reductases, this column procedure should be applicable to purification of dihydropteridine reductase from brain tissue. Properties Dihydropterin reductase isolated from sheep brain is similar in most respects to other dihydropterin reductases such as that from sheep liver~,l° and from beef liver.ll The sheep brain reductase has a pH optimum of approximately 7, similar to the reductase from liver. 6 It is markedly inhibited by thiol reagents such as HgCI2 and p-chloromercuribenzoate (pCMB). For exam9 M. M. Chauvin, K. K. Korri, A. Tirpak, R. C. Simpson, and K. G. Scrimgeour, Can. J. Biochem. 57, 178 (1979). J0 j. E. Craine, E. S. Hall, and S. Kaufman, J. Biol. Chem. 247, 6082 (1972). " K. K. Korri, D. Chippel, M. M. Chauvin, A. Tirpak, and K. G. Scrimgeour, Can. J. Biochem. 55, 1145 (1977).
12.7 8.7 4.9
132
CATABOLISM OF THE AROMATIC AMINO ACIDS
[18]
pie, incubation of the enzyme for 3 min with approximately 2/~M HgCI2 or 200 /.~M pCMB decreased the activity of the reductase by one-half. Addition of N A D H to the enzyme before incubation with the thiol reagent prevented inactivation. Iodoacetic acid at concentrations as high as 3 × 10 -3 M gave no observable inhibition. From these results, it would appear that the reductase has a thiol group at or near its active site. The reductase from brain demonstrated hyperbolic kinetics for both the quinonoid dihydropterin and NADH. The Km for quinonoid dihydro6,7-dimethyldihydropterin is 1.0 × 10-5 M and that for NADH is 2.2 × 10-5 M. These values are similar to those determined for the reductase from sheep liver. 6 NAD + is an inhibitor that is competitive with NADH. When the reductase was applied to a column of Sephadex G-200, it eluted at the position expected for a protein having a molecular weight of approximately 47,000. It has a mobility in SDS gels that corresponds to a molecular weight of 27,000. The liver enzyme gave two protein bands with molecular weights of about 27,000 and 55,000 when it was treated with the cross-linking compound dimethylsuberimidate 6 and analyzed by SDS electrophoresis. We have concluded that the reductase from sheep brain is a dimer of identical protomeric chains, just as is the sheep liver reductase. 6 In addition to these similarities in molecular weight, the amino acid compositions of the brain and liver enzymes were found to be quite similar. 6
[18] 4 - H y d r o x y p h e n y l p y r u v a t e
Dioxygenase from Pig Liver
By DAVID J. BUCKTHAL, PATRICK A. ROCHE, THOMAS J. MOOREHEAD, BRIAN J. R. FORBES, and GOROON A. HAMILTON O
/CH2COO-
oo o Ho@o
4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.22), an enzyme that participates in the catabolism of tyrosine in most organisms, is found in the liver and kidney of mammals. The procedure given here for its purification from pig liver differs only slightly from that reported previously, i P. A. Roche, T. J. Moorehead, and G. A. Hamilton, Arch. Biochem. Biophys. 216, 62 (1982).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
DIHYDROPTERIDINE REDUCTASE FROM SHEEP BRAIN
127
[17] D i h y d r o p t e r i d i n e R e d u c t a s e f r o m S h e e p B r a i n
By K. G. SCRIMGEOURand S. CHEEMA-DHADLI Introduction Dihydropterin reductase catalyzes the reduction of the unstable quinonoid dihydropterin formed during pterin-requiring hydroxylation reactions. Because NADH is the preferred reductant, the systematic name for the enzyme is NADH:6,7-dihydropteridine oxidoreductase (EC 1.6.99.7); however, it usually is called either dihydropterin or dihydropteridine reductase. Because tyrosine hydroxylase L2 and tryptophan hydroxylase 3-5 activities had been detected in brain tissue, we tested sheep brain as a potential source of the supplementary enzyme dihydropterin reductase, required for aromatic hydroxylations. The reductase was purified to homogeneity using a nine-step purification procedure, and the purified enzyme was compared to the dihydropterin reductase from sheep liver. 6 Quinonoid dihydropterin + N A D H + H + ~ 5,6,7,8-tetrahydropterin + N A D +
Assay Method
Principle Dihydropterin reductase activity was routinely measured by following the decrease in absorbance at 340 nm corresponding to oxidation of NADH. Quinonoid dihydropterin was prepared in situ in the reaction mixture from 6,7-dimethyltetrahydropterin, by oxidation with a stoichiometric amount of dichlorophenolindophenol. The quinonoid dihydropterin is then reduced back to the tetrahydropterin by NADH (chemical reaction) and by NADH in the presence of the reductase (enzymatic reaction). i T. N a g a t s u , M. Levitt, and S. Udenfriend, Biochem. Biophys. Res. Commun. 14, 543 (1964). 2 S. F a h n , J. S. R o d m a n , and L. J. Cote, J. Neurochem. 16, 1293 (1969). 3 D. G. G r a h a m e - S m i t h , Biochem. J. 105, 351 (1967). 4 D. A. V. Peters, P. L. McGeer, and E. C. McGeer, J. Neurochem. 15, 1431 (1968). 5 A. I c h i y a m a , S. N a k a m u r a , Y. N i s h i z u m a , and O. Hayaishi, J. Biol. Chem. 245, 1699 (1970). 6 S. C h e e m a , S. J. Soldin, A. K n a p p , T. H o f m a n n , and K. G. Scrimgeour, Can. J. Biochem. 51, 1229 (1973).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
128
CATABOLISM OF THE AROMATIC AMINO ACIDS
[17]
Reagents 6,7-Dimethyl-5,6,7,8-tetrahydropterin hydrochloride, 1.5 mM Dichlorophenolindophenol, 1.5 mM Tris-HC1, pH 6.8, 0.1 M NADH, 2 mM All reagents listed above were kept deaerated by bubbling with moist nitrogen gas. Procedure The reaction mixture contained 0.9 ml of Tris buffer, 0.05 ml of 6,7dimethyltetrahydropterin, 0.05 ml of NADH, 0.05 ml of dichlorophenolindophenol, and enough enzyme (e.g., 0.005 ml) to give a change in absorbance at 340 nm of 0.1 to 0.2/min. The enzyme was omitted from the reference cell. The reaction was initiated by the addition of the indophenol to both the experimental and reference cuvettes. The change in absorbance was recorded using a recording spectrophotometer with a scale expander and a constant temperature cell holder maintained at 25° using a thermostatted water bath. The reference cell contained all the components except the enzyme, and so provided the correction for the chemical reaction. For kinetic measurements, the initial velocity of each reaction was measured over an initial 10 to 20 sec, and a unit of activity was taken as that amount of enzyme that gave a change in absorbance of 0.62/ml/min due to the oxidation of NADH to NAD ÷. This is equivalent to the oxidation of approximately 0.1 /xmol of NADH. All assays were done in at least duplicate. Under the conditions of our assay, the oxidation of NADH by dichlorophenolindophenol was insignificant, so it was not necessary to apply the elaborate correction calculations used by Nielsen et al. 7 The reaction rates were linear over the time period used, and the initial velocities were directly proportional to the concentration of enzyme added. Purification Where possible, all manipulations were performed at 0-4 °. Frozen sheep brains (700 g) were partially thawed, and chopped into small pieces. The pieces were homogenized for 60 sec with 2 liters of cold 0.25 M sucrose in a Waring blender at one-third the full speed of the blender. The homogenate was centrifuged at 5000 rpm in a Lourdes Betafuge (4-liter head) for 20 min. The residue was mixed with another 1.5 liters of su7 K. H. Nielsen, V. S i m o n s e n , and K. E. Lind, Eur. J. Biochem. 9, 497 (1969).
[17]
D I H Y D R O P T E R I D I N E R E D U C T A S E F R O M S H E E P BRAIN
129
crose solution and homogenized again for another 20 sec. The second homogenate was then centrifuged and the two supernatant solutions were pooled. Solid ammonium sulfate was slowly stirred into the solution from the previous step until the final concentration of ammonium sulfate was 25%. This suspension was centrifuged and the residue was discarded. More ammonium sulfate was added to the supernatant fraction until 70% saturation was reached. The precipitate was collected by centrifugation and dissolved in about one-seventh the original volume of 0.02 M Tris buffer, pH 7.4 containing 10-3 M 2-mercaptoethanol, and dialyzed overnight against a large volume of the same buffer. [Mercaptoethanol (10 -3 M) was included in all buffers used in the purification.] Drops of zinc acetate (0.2 M) were stirred into the dialyzed solution until the ratio of zinc acetate solution to enzyme solution was 1 : 10. The suspension was stirred for 30 min until there was a thick cloudy precipitate, which was removed by centrifugation. The supernatant fraction was dialyzed overnight against two changes of 0.05 M Tris buffer, pH 7.4. To this dialyzed solution, solid ammonium sulfate (10 g/100 ml of solution) was added, and the pH of the solution was adjusted to 8.2 with 1 N ammonium hydroxide. Another 7 g of solid ammonium sulfate/100 ml of solution was added slowly. The precipitate was removed by centrifugation and discarded. After another 30 g of ammonium sulfate/100 ml of solution had been added, the solution was again centrifuged and the precipitate was dissolved in one-third the original volume of 0.01 M Tris buffer. To this solution, 0.2 M zinc acetate (4 ml/100 ml of protein solution) was added. A cloudy precipitate was seen after a few minutes and the solution was centrifuged. The supernatant fraction was dialyzed against 15 times its volume of 0.01 M Tris buffer, pH 7.4. Calcium phosphate gel (52.5 mg solid/ml) was added dropwise in the ratio of 20 ml of gel to 100 ml of the previous solution. The mixture was stirred for 30 min, centrifuged, and the supernatant fraction discarded. The gel was washed with 0.008 M phosphate, pH 6.8, and the supernatant solution again discarded. The gel was further washed with 0.1 M phosphate, pH 6.8, using a volume two-thirds that of the untreated enzyme solution. The supernatant solution obtained by centrifugation was dialyzed against a large volume of 0.03 M Tris buffer, pH 7.5. A column (2.5 x 40 cm) was packed with DEAE-Sephadex A-50 equilibrated with 0.03 M Tris buffer, pH 7.5. A dialyzed solution of enzyme (180 ml, 0.98 mg/ml) was applied to the column and eluted with a gradient of 500 ml of 0.05 M Tris buffer, in the mixing flask, and 500 ml of 0.3 M Tris, pH 7.5 in the reservoir. The enzyme was eluted at about 0.2 M buffer concentration with about a 10-fold purification.
130
CATABOLISM OF THE AROMATIC AMINO ACIDS
[17]
The fractions from the DEAE-Sephadex column containing enzyme activity were pooled and concentrated about 10-fold by ultrafiltration in a Diaflo apparatus. A column (1.5 x 90 cm) was packed with Sephadex G200 and equilibrated with 0.05 M Tris buffer, pH 7.5. The enzyme sample was applied in and eluted with 0.05 M Tris at 6 ml/hr. Of the two major protein peaks eluted, the second peak contained the enzyme activity. Those fractions containing activity were pooled and again concentrated in the Diaflo. Finally, the enzyme was purified by preparative polyacrylamide electrophoresis. The gel (5% acrylamide, 0.13% bisacrylamide, 0.007% fresh ammonium persulfate) was cast in a Pyrex glass tube (1.5 x 15 cm) open at both ends. After it was polymerized at room temperature, a 1 cm stacking gel was polymerized at the top of the column. 8 Ice-cold 0.03 M Tris-glycine buffer, pH 8.3, containing 10 -3 M 2-mercaptoethanol was used in both reservoirs and electrophoresis was performed at 0 to 4 ° at a constant current of 5 mA. Before application of the enzyme sample, electrophoresis of 0.5 ml of 0.002% bromphenol blue, which contained two or three drops of 40% sucrose, was carried out for 1 hr. This was to avoid mixing the enzyme sample with the tracking dye and also to get rid of the ammonium persulfate from the gel. 2-Mercaptoethanol (5/xl) and two or three drops of 40% sucrose were added to the enzyme sample before its application. The electrophoresis was carried out for 8 hr. During the electrophoresis, 5-/xl samples of 2-mercaptoethanol were added two or three times to the top buffer reservoir, to maintain the activity of the enzyme. At the end of the electrophoretic run, the gel was carefully removed from the tube and sliced into 1-2 mm slices. The slices were broken up by suspension in 2 ml of 0.05 M Tris buffer, pH 7.3, overnight at 4 °. The protein concentration of the supernatant solution was determined from its absorbance at 280 nm, and the enzyme activity was determined on 2-/zl aliquots. The gel slices containing the enzyme activity were extracted once or twice more. Usually only two slices contained most of the activity. The supernatant solutions from these were pooled and concentrated. The results of the purification procedure are summarized in Table I. The specific activity of a typical preparation was 61 units/mg, with an overall yield of about 5%. The sheep brain reductase was shown to be pure by analytical disc gel electrophoresis. The one band of protein coincided with activity of the enzyme, when activity was determined on slices of unstained gel. 8 B. J. Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).
[17]
DIHYDROPTERIDINE REDUCTASE FROM SHEEP BRAIN
131
TABLE 1 SUMMARY OF THE PURIFICATION OF qDHP REDUCTASE FROM SHEEP BRAIN"
Specific activity Step 1. Extract 2. Ammonium
Total volume (ml)
Total
Total
activity
protein
Yield
(units)
(mg)
(%)
0.9 !.5
3200 860
35,200 20,600
38,400 13,700
100 60
16.6 28.9
2.8 3.6
880 350
14,600 10,100
5,190 2,800
42 30
5.4
28
5.2
360
10,000
1,940
30
2.6
35.2
14
175
6,160
0.26 0.4 0.08
37.8 123 49.2
145 307 610
118 25 35
4,450 3,080 1,724
Protein
Number of
(mg/ml)
units/ml
12.6 16.4
11 24.1
5.9 8.0
(unit/mg protein)
sulfate 3. First zinc acetate 4. Alkaline ammonium sulfate 5. Second zinc
acetate 6. Calcium phosphate eluate 0.1 M
455
14.7
phosphate 7. DEAE-Sephadex 8. Sephadex G-200 9. Preparative disc
30.6 10 2.8
electrophoresis " Frozen sheep brain (700g).
Dihydropteridine reductase from beef kidney has been purified using immobilized Cibacron Blue. 9 Because of the similarities of brain and sheep reductases, this column procedure should be applicable to purification of dihydropteridine reductase from brain tissue. Properties Dihydropterin reductase isolated from sheep brain is similar in most respects to other dihydropterin reductases such as that from sheep liver~,l° and from beef liver.ll The sheep brain reductase has a pH optimum of approximately 7, similar to the reductase from liver. 6 It is markedly inhibited by thiol reagents such as HgCI2 and p-chloromercuribenzoate (pCMB). For exam9 M. M. Chauvin, K. K. Korri, A. Tirpak, R. C. Simpson, and K. G. Scrimgeour, Can. J. Biochem. 57, 178 (1979). J0 j. E. Craine, E. S. Hall, and S. Kaufman, J. Biol. Chem. 247, 6082 (1972). " K. K. Korri, D. Chippel, M. M. Chauvin, A. Tirpak, and K. G. Scrimgeour, Can. J. Biochem. 55, 1145 (1977).
12.7 8.7 4.9
132
CATABOLISM OF THE AROMATIC AMINO ACIDS
[18]
pie, incubation of the enzyme for 3 min with approximately 2/~M HgCI2 or 200 /.~M pCMB decreased the activity of the reductase by one-half. Addition of N A D H to the enzyme before incubation with the thiol reagent prevented inactivation. Iodoacetic acid at concentrations as high as 3 × 10 -3 M gave no observable inhibition. From these results, it would appear that the reductase has a thiol group at or near its active site. The reductase from brain demonstrated hyperbolic kinetics for both the quinonoid dihydropterin and NADH. The Km for quinonoid dihydro6,7-dimethyldihydropterin is 1.0 × 10-5 M and that for NADH is 2.2 × 10-5 M. These values are similar to those determined for the reductase from sheep liver. 6 NAD + is an inhibitor that is competitive with NADH. When the reductase was applied to a column of Sephadex G-200, it eluted at the position expected for a protein having a molecular weight of approximately 47,000. It has a mobility in SDS gels that corresponds to a molecular weight of 27,000. The liver enzyme gave two protein bands with molecular weights of about 27,000 and 55,000 when it was treated with the cross-linking compound dimethylsuberimidate 6 and analyzed by SDS electrophoresis. We have concluded that the reductase from sheep brain is a dimer of identical protomeric chains, just as is the sheep liver reductase. 6 In addition to these similarities in molecular weight, the amino acid compositions of the brain and liver enzymes were found to be quite similar. 6
[18] 4 - H y d r o x y p h e n y l p y r u v a t e
Dioxygenase from Pig Liver
By DAVID J. BUCKTHAL, PATRICK A. ROCHE, THOMAS J. MOOREHEAD, BRIAN J. R. FORBES, and GOROON A. HAMILTON O
/CH2COO-
oo o Ho@o
4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.22), an enzyme that participates in the catabolism of tyrosine in most organisms, is found in the liver and kidney of mammals. The procedure given here for its purification from pig liver differs only slightly from that reported previously, i P. A. Roche, T. J. Moorehead, and G. A. Hamilton, Arch. Biochem. Biophys. 216, 62 (1982).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.