442
PYRIMIDINE METABOLIZING ENZYMES
[59]
Stability. During the latter states of purification thymidine phosphorylase is quite labile. The use of 2-mercaptoethanol and sucrose during precipitation procedures with ammonium sulfate will stabilize the enzyme. Purified thymidine phosphorylase can be stored in 10 mM potassium phosphate, pH 7.5, 10 mM in 2-mercaptoethanol and containing 20% sucrose for 3 months at 4 ° without loss of enzyme activity. Effect ofpH. Optimum activity is found at pH 7.5-8.0 with a rapid decrease in activity both above and below this range.
[59] T h y m i d i n e
Phosphorylase
f r o m Escherichia coli
By MARIANNE SCHWARTZ T h y m i d i n e + p h o s p h a t e ,~, t h y m i n e + d e o x y r i b o s e - l - p h o s p h a t e
Assay of Enzymic Activity
Principle. The assay used for following the purification of thymidine phosphorylase is based on the difference in molar extinction coefficient between thymidine and thymine at alkaline pH. 1 At 300 nm this difference is 3.4 × 103. For more accurate activity determinations the diphenylamine assay is employed. 2 Both assays can be used on crude extracts. Reagents Thymidine 0.1 M K-phosphate, 0.1 M, pH 7.1 Buffer: Tris'HC1 buffer, 10 mM, pH 7.3, is made 2 mM with respect to EDTA NaOH, 0.5 N
Procedure. Buffer and enzyme in a total volume of 0.850 ml are mixed at 0 °. Then 0.1 ml K-phosphate is added and the mixture transferred to 37 ° and incubated for 2 min. The reaction is started by addition of 0.05 ml thymidine. At 5-min time intervals 0.300-ml samples are transferred to 0.700-ml NaOH. The amount of thymine formed is measured at 300 nm. If the 1R. D. H o t c h k i s s , J. Biol. Chem. 175, 315 (1948). 2K. Burton, Biochem. J. 62, 315 (1956).
M E T H O D S IN E N Z Y M O L O G Y , V O L . LI
Copyright © 1978by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
[59]
THYMIDINE PHOSPHORYLASE
443
diphenylamine method is employed the reaction is stopped by transferring the samples to tubes containing O. 150 ml of 30% perchloric acid.
Definition of Unit and Specific Activity. One unit is defined as the amount of enzyme that cleaves 1 /zmole of thymidine in 1 min at 37 °. Specific activity is units per milligram of protein. Protein is determined by the method of Lowry et al. 3 Purification Procedure
Bacterial Strains and Growth of Cells. E. coli K-12 (thyA-, deoC-, met-) is used for the purification described below, taking advantage of the high specific activity obtained following thymine starvation. Cells are grown in a glucose minimal medium with addition of 0.016 mM thymine and 0.05 mg/ml methionine. At an absorbance at 450 nm of about 1.0, 2 liters of culture are diluted into 9 liters of prewarmed medium without thymine. The resulting thymine concentration should be less than 3/Z~/. After 8 hr of thymine starvation the absorbance at 450 nm is about 2.0. The cells are then harvested by centrifugation. The cells are washed in 0.9% NaC1 and stored at - 6 0 °. E. coil Strains Constitutive for Thymidine Phosphorylase (deoRC). By employing these strains thymine starvation can be avoided. Twohundred-milliliter overnight cultures in glycerol minimal medium are used as inoculum for 5-liter flasks each containing 1.8 liters of prewarmed glycerol minimal medium. The flasks are incubated with shaking at 37 ° overnight. The cells are harvested as described above. The use of deoR ~ strains results in the same specific activity in crude extracts as thymine starvation, i.e., about 100 times the wild-type level. The purification procedure is identical for both strains. Step 1. Disruption of Cells. The cell pellet is thawed and suspended in 2.5 volumes of 0.01 M Tris-HC1, 2 mM EDTA, pH 7.9. The cells are broken by sonic treatment or by using a French press. The cell debris is removed by centrifugation in a refrigerated Sorvall centrifuge at 40,000 g for 60 rain. Step 2. Streptomycin Sulfate Precipitation. To each milliliter of the supernatant fluid 0.23 ml of 10% streptomycin sulfate is added over a period of 30 min to a final concentration of 1.9%. After another 30 rain the precipitate is removed by centrifugation, and the pH of the supernatant fluid is adjusted to 7.8 by addition of 1.0 M Tris-HCl, pH 10.0. 30. H. Lowry, N. J. Rosenbrough, and R. J. Randall, J. Biol. Chem. 193, 265 (1961).
444
PYR1M1DINE METABOLIZING ENZYMES
[59]
Step 3. Ammonium Sulfate Fractionation. The streptomycin sulfate supernatant fluid is treated with solid ammonium sulfate (260 mg/ml) to 0.43 saturation. After standing 30 min at 0 °, the suspension is centrifuged and the pellet discarded. Solid ammonium sulfate (205 mg/ml) is further added to 0.73 saturation to precipitate the enzyme. After centrifugation the precipitate is dissolved in 0.i M Tris'HCI-2 mM EDTA (pH 7.6) to a total volume equal to one-tenth of the original extract. Step 4. Sephadex G-IO0 Chromatography. The dissolved precipitate from ammonium sulfate fractionation is then directly applied on a G-100 Sephadex column (2.5 × 90 cm), equilibrated with 10 mM Trissuccinate-50 mM NaCI-1 mM EDTA (pH 6.8). Flow rate is maintained at 18 ml/hr. Fractions of 3 ml are collected, and those containing the enzyme are pooled. Step 5. DEAE-Sephadex Chromatography. 4 The pooled fractions from step 4 are applied directly on a 2.5 × 40 cm DEAE-Sephadex column, equilibrated with the same buffer as the G-100 column. The column is eluted with a linear gradient from 50 mM NaCI to 300 mM NaCI in 10 mM Tris-succinate-1 mM EDTA (pH 6.8). The total gradient volume is 2.5 liters. Flow rate is maintained at 20 ml/hr. The fractions containing the enzyme are pooled. The enzyme is eluted at a concentration of about 150 mM NaC1. Step 6. Hydroxyapatite Chromatography. The pooled fractions are concentrated 5- to 10-fold by pressure filtration (Amicon PQM 10 filter) and dialyzed overnight against 20 mM K-phosphate, pH 6.8. The dialyzed solution is applied on a 1.5 x 30 cm hydroxyapatite column, equilibrated with the above buffer. Thymidine phosphorylase is eluted with a linear gradient of K-phosphate buffer from 20-150 mM, pH 6.8. Total gradient volume is 600 ml. Flow rate is maintained at 10 ml/hr. Fractions containing the enzyme are collected. The enzyme is eluted at a phosphate concentration of approximately 100 mM. The purification procedure is summarized in the table) Properties
Specificity. Thymidine phosphorylase is specific for pyrimidine deoxyribonucleosides. Purine deoxyribonucleosides and ribonucleosides as DEAE-cellulose DE-52 gives identical elution profiles. ~A. Munch-Petersen, P. Nygaard, K. Hammer-Jespersen, and N. Fill, Eur. J. Biochem. 27, 208 (1972).
[59]
445
THYMIDINE PHOSPHORYLASE
PURIFICATION PROCEDURE FOR THYMIDINE PHOSPHORYLASEFROM Escherichia col[ '1
Volume
Specific activity
Fraction
(ml)
(units/rag)
Recovery (%)
Sonic extract Streptomycin supernatant Ammonium sulfate precipitate G-100 eluate
65 77 13 72 250
5.22 3.85 7.02 11.95 28.7
100 73 68 51 38
DEAE-sephadex
(not concentrated) Hydroxyapatite
87
122
36
Data given are from a purification starting with 20 g of cells ofE. coli So 103 (thyA(deoR c) are available from this laboratory or can be selected by the procedure described by Munch-
deoC-, met-). Strains constitutive for thymidine phosphorylase
Petersen et al. 5
well as deoxycytidine are not cleaved by the enzyme. Arsenate can replace phosphate.
Stability. The enzyme is stable for several months when kept in 10 mM Tris.HC1, pH 7.3, at - 2 0 °. At 4 ° the activity is gradually lost. The stability is dependent on the protein concentration. Effect of pH. The enzyme shows a rather broad pH dependency, with maximum activity at pH 6.3. Kinetic Constants. The enzyme shows Michaelis-Menten kinetics. Initial velocity studies indicate an ordered sequential reaction mechanism. 6 Phosphate isthe first substrate to bind to and deoxyribose the last product to dissociate from the enzyme. Kin: phosphate 0.89 raM; thymidine 0.38 mM. The enzyme is competitively inhibited by ribose-1phosphate. Molecular Weight. The enzyme has a molecular weight of 90,000 r as determined by G-200 gel filtration. The enzyme seems to consist of two identical subunits showed by SDS polyacrylamide electrophoresis after intensive boiling with SDS. 8
~M. Schwartz, Eur. J. Biochem. 21, 191 (1971). 7This is a correction of the earlier published value. 6 M. Schwartz, unpublished results. 8 B. Svenningsen, unpublished results.
PYRIMIDINE METABOLIZING ENZYMES
[59]
Stability. During the latter states of purification thymidine phosphorylase is quite labile. The use of 2-mercaptoethanol and sucrose during precipitation procedures with ammonium sulfate will stabilize the enzyme. Purified thymidine phosphorylase can be stored in 10 mM potassium phosphate, pH 7.5, 10 mM in 2-mercaptoethanol and containing 20% sucrose for 3 months at 4 ° without loss of enzyme activity. Effect ofpH. Optimum activity is found at pH 7.5-8.0 with a rapid decrease in activity both above and below this range.
[59] T h y m i d i n e
Phosphorylase
f r o m Escherichia coli
By MARIANNE SCHWARTZ T h y m i d i n e + p h o s p h a t e ,~, t h y m i n e + d e o x y r i b o s e - l - p h o s p h a t e
Assay of Enzymic Activity
Principle. The assay used for following the purification of thymidine phosphorylase is based on the difference in molar extinction coefficient between thymidine and thymine at alkaline pH. 1 At 300 nm this difference is 3.4 × 103. For more accurate activity determinations the diphenylamine assay is employed. 2 Both assays can be used on crude extracts. Reagents Thymidine 0.1 M K-phosphate, 0.1 M, pH 7.1 Buffer: Tris'HC1 buffer, 10 mM, pH 7.3, is made 2 mM with respect to EDTA NaOH, 0.5 N
Procedure. Buffer and enzyme in a total volume of 0.850 ml are mixed at 0 °. Then 0.1 ml K-phosphate is added and the mixture transferred to 37 ° and incubated for 2 min. The reaction is started by addition of 0.05 ml thymidine. At 5-min time intervals 0.300-ml samples are transferred to 0.700-ml NaOH. The amount of thymine formed is measured at 300 nm. If the 1R. D. H o t c h k i s s , J. Biol. Chem. 175, 315 (1948). 2K. Burton, Biochem. J. 62, 315 (1956).
M E T H O D S IN E N Z Y M O L O G Y , V O L . LI
Copyright © 1978by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
[59]
THYMIDINE PHOSPHORYLASE
443
diphenylamine method is employed the reaction is stopped by transferring the samples to tubes containing O. 150 ml of 30% perchloric acid.
Definition of Unit and Specific Activity. One unit is defined as the amount of enzyme that cleaves 1 /zmole of thymidine in 1 min at 37 °. Specific activity is units per milligram of protein. Protein is determined by the method of Lowry et al. 3 Purification Procedure
Bacterial Strains and Growth of Cells. E. coli K-12 (thyA-, deoC-, met-) is used for the purification described below, taking advantage of the high specific activity obtained following thymine starvation. Cells are grown in a glucose minimal medium with addition of 0.016 mM thymine and 0.05 mg/ml methionine. At an absorbance at 450 nm of about 1.0, 2 liters of culture are diluted into 9 liters of prewarmed medium without thymine. The resulting thymine concentration should be less than 3/Z~/. After 8 hr of thymine starvation the absorbance at 450 nm is about 2.0. The cells are then harvested by centrifugation. The cells are washed in 0.9% NaC1 and stored at - 6 0 °. E. coil Strains Constitutive for Thymidine Phosphorylase (deoRC). By employing these strains thymine starvation can be avoided. Twohundred-milliliter overnight cultures in glycerol minimal medium are used as inoculum for 5-liter flasks each containing 1.8 liters of prewarmed glycerol minimal medium. The flasks are incubated with shaking at 37 ° overnight. The cells are harvested as described above. The use of deoR ~ strains results in the same specific activity in crude extracts as thymine starvation, i.e., about 100 times the wild-type level. The purification procedure is identical for both strains. Step 1. Disruption of Cells. The cell pellet is thawed and suspended in 2.5 volumes of 0.01 M Tris-HC1, 2 mM EDTA, pH 7.9. The cells are broken by sonic treatment or by using a French press. The cell debris is removed by centrifugation in a refrigerated Sorvall centrifuge at 40,000 g for 60 rain. Step 2. Streptomycin Sulfate Precipitation. To each milliliter of the supernatant fluid 0.23 ml of 10% streptomycin sulfate is added over a period of 30 min to a final concentration of 1.9%. After another 30 rain the precipitate is removed by centrifugation, and the pH of the supernatant fluid is adjusted to 7.8 by addition of 1.0 M Tris-HCl, pH 10.0. 30. H. Lowry, N. J. Rosenbrough, and R. J. Randall, J. Biol. Chem. 193, 265 (1961).
444
PYR1M1DINE METABOLIZING ENZYMES
[59]
Step 3. Ammonium Sulfate Fractionation. The streptomycin sulfate supernatant fluid is treated with solid ammonium sulfate (260 mg/ml) to 0.43 saturation. After standing 30 min at 0 °, the suspension is centrifuged and the pellet discarded. Solid ammonium sulfate (205 mg/ml) is further added to 0.73 saturation to precipitate the enzyme. After centrifugation the precipitate is dissolved in 0.i M Tris'HCI-2 mM EDTA (pH 7.6) to a total volume equal to one-tenth of the original extract. Step 4. Sephadex G-IO0 Chromatography. The dissolved precipitate from ammonium sulfate fractionation is then directly applied on a G-100 Sephadex column (2.5 × 90 cm), equilibrated with 10 mM Trissuccinate-50 mM NaCI-1 mM EDTA (pH 6.8). Flow rate is maintained at 18 ml/hr. Fractions of 3 ml are collected, and those containing the enzyme are pooled. Step 5. DEAE-Sephadex Chromatography. 4 The pooled fractions from step 4 are applied directly on a 2.5 × 40 cm DEAE-Sephadex column, equilibrated with the same buffer as the G-100 column. The column is eluted with a linear gradient from 50 mM NaCI to 300 mM NaCI in 10 mM Tris-succinate-1 mM EDTA (pH 6.8). The total gradient volume is 2.5 liters. Flow rate is maintained at 20 ml/hr. The fractions containing the enzyme are pooled. The enzyme is eluted at a concentration of about 150 mM NaC1. Step 6. Hydroxyapatite Chromatography. The pooled fractions are concentrated 5- to 10-fold by pressure filtration (Amicon PQM 10 filter) and dialyzed overnight against 20 mM K-phosphate, pH 6.8. The dialyzed solution is applied on a 1.5 x 30 cm hydroxyapatite column, equilibrated with the above buffer. Thymidine phosphorylase is eluted with a linear gradient of K-phosphate buffer from 20-150 mM, pH 6.8. Total gradient volume is 600 ml. Flow rate is maintained at 10 ml/hr. Fractions containing the enzyme are collected. The enzyme is eluted at a phosphate concentration of approximately 100 mM. The purification procedure is summarized in the table) Properties
Specificity. Thymidine phosphorylase is specific for pyrimidine deoxyribonucleosides. Purine deoxyribonucleosides and ribonucleosides as DEAE-cellulose DE-52 gives identical elution profiles. ~A. Munch-Petersen, P. Nygaard, K. Hammer-Jespersen, and N. Fill, Eur. J. Biochem. 27, 208 (1972).
[59]
445
THYMIDINE PHOSPHORYLASE
PURIFICATION PROCEDURE FOR THYMIDINE PHOSPHORYLASEFROM Escherichia col[ '1
Volume
Specific activity
Fraction
(ml)
(units/rag)
Recovery (%)
Sonic extract Streptomycin supernatant Ammonium sulfate precipitate G-100 eluate
65 77 13 72 250
5.22 3.85 7.02 11.95 28.7
100 73 68 51 38
DEAE-sephadex
(not concentrated) Hydroxyapatite
87
122
36
Data given are from a purification starting with 20 g of cells ofE. coli So 103 (thyA(deoR c) are available from this laboratory or can be selected by the procedure described by Munch-
deoC-, met-). Strains constitutive for thymidine phosphorylase
Petersen et al. 5
well as deoxycytidine are not cleaved by the enzyme. Arsenate can replace phosphate.
Stability. The enzyme is stable for several months when kept in 10 mM Tris.HC1, pH 7.3, at - 2 0 °. At 4 ° the activity is gradually lost. The stability is dependent on the protein concentration. Effect of pH. The enzyme shows a rather broad pH dependency, with maximum activity at pH 6.3. Kinetic Constants. The enzyme shows Michaelis-Menten kinetics. Initial velocity studies indicate an ordered sequential reaction mechanism. 6 Phosphate isthe first substrate to bind to and deoxyribose the last product to dissociate from the enzyme. Kin: phosphate 0.89 raM; thymidine 0.38 mM. The enzyme is competitively inhibited by ribose-1phosphate. Molecular Weight. The enzyme has a molecular weight of 90,000 r as determined by G-200 gel filtration. The enzyme seems to consist of two identical subunits showed by SDS polyacrylamide electrophoresis after intensive boiling with SDS. 8
~M. Schwartz, Eur. J. Biochem. 21, 191 (1971). 7This is a correction of the earlier published value. 6 M. Schwartz, unpublished results. 8 B. Svenningsen, unpublished results.
