[58]
THYMIDINE PHOSPHORYLASE
437
at pH 7.4. Fraction 6 was used as enzyme; the measured Vmax with uridine as substrate was 63.5 /zmoles/min/mg of protein. The Km for Pi, determined with uridine (1.2 mM) as substrate, was 0.38 mM. Uridine concentrations above 15 mM were somewhat inhibitory; no other nucleoside substrate tested had this effect. The conclusion that a single pyrimidine nucleoside phosphorylase cleaves both uridine and thymidine in H. influenzae rests on several lines of evidence. First, the two activities maintained a constant ratio over a 400-fold purification and were not resolved by analytical polyacrylamide gel electrophoresis. Second, the activities showed identical kinetics of heat inactivation and were identically protected against heating by several nucleoside substrates. Third, kinetic studies with ribo- and deoxynucleoside substrate pairs gave results which conformed quantitatively with expectation for a single enzyme comprising both activities. 2 The pattern of substrate specificity observed is quite similar to that of the uridine phosphorylase purified from several mammalian sources, which were observed to retain significant activity toward deoxynucleosides. 5-g Bacillus stearothermophilus was found to possess a single pyrimidine nucleoside phosphorylase which was 1.5 times as active with thymidine as with uridinef this ratio for the H. influenzae enzyme was 0.25. The enzyme described here shows a striking preference for 5bromodeoxyuridine as a substrate; the initial rate observed with this substrate was 5 times that found with any other deoxynucleoside. ST. 6 E. 7A. 8 H. aP.
A. Krenitsky, M. Barclay, and J. A. Jacquez, J. Biol. Chem. 239, 805 (1964). W. Yamada, J. Biol. Chem. 243, 1649 (1968). Kraut and E. W. Yamada, J. Biol. Chem. 246, 2021 (1971). Pontis, G. Degerstedt, and P. Reichard, Biochim. Biophys. Acta 51, 139 (1961). P. Saunders, B. A. Wilson, and G. F. Saunders, J. Biol. Chem. 244, 3691 (1969).
[58] T h y m i d i n e
from Salmonella
Phosphorylase
typhim urium B y PATRICIA A . H O F F E E a n d JAMES B L A N K Thymidine + P~ ~ thymine + 2-deoxy-D-ribose-l-phosphate
In Escherichia coli and Salmonella typhimurium, thymidine phosphorylase plays an important role in the metabolism of thymine auxotrophs and is necessary for the conversion of exogenous thymine to thymidine. In low-thymine-requiring strains, thymidine phosphorylase becomes METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181951-5
438
PYRIMIDINE METABOLIZINGENZYMES
[58]
phenotypically constitutive in strains containing a mutation in deoxyribose-5-phosphate aldolase. 1-a Thymidine phosphorylase is regulated along with deoxyribose-5-phosphate aldolase, phosphodeoxyribomutase, and purine nucleoside phosphorylase as part of the deo regulon. 2-a
Assay Method 1° Principle. Enzyme activity is measured spectrophotometrically by measuring the formation of thymine from thymidine in the presence of sodium arsenate. Reagents Tris (hydroxymethyl) aminomethane-HC1 buffer 0.2 M, pH 7.4, with 0.1 M sodium arsenate Thymidine, 4 mg dissolved in 1 ml of the Tris.HC1 buffer NaOH 0.3 N Procedure. Add 0.2 ml of the thymidine-buffer solution to 3 tubes marked 0, 5, and 10 min. Add enzyme solution at 0°. To the 0 time tube add 1 ml of NaOH and incubate the 5- and 10-min tubes at 37°. Stop the reaction at the specified time by adding 1 ml of 0.3 N NaOH. The optical density of each sample is then determined in a spectrophotometer set at 300 nm. The increase in optical density is plotted against time. Definition o f Units. One unit of activity is defined as the number of /zmoles of thymine produced per minute at 37°. An extinction coefficient of 4.04 is used. Protein is determined by the method of Lowry et al. 11 before ammonium sulfate fractionation (see Purification) and by the method of B/icher 12after ammonium sulfate fractionation. 1W. E. Razzell and G. G. Khorana, Biochim. Biophys. Acta 28, 562 (1958). ~T. R. Breitman and R. M. Bradford, Biochim. Biophys. Acta 138, 217 (1967). aP. Hoffee, J. Bacteriol. 95, 449 (1968). 4A. Munch-Peterson. Fur. J. Biochem. 6, 432 (1968). ~M. S. Lomax and G. R. Greenberg. J. Bacteriol. 96, 501 (1968). 6p. Hoffee and B. C. Robertson, J. Bacteriol. 97, 1386 (1969). 7B. C. Robertson, P. Jargiello, J. Blank, and P. Hoffee, J. Baeteriol. 102, 628 (1970). s S. Ahmad and R. H. Pritchard, Mol. Gen. Genet. 104, 351 (1969). aJ. Blank and P. Hoffee, Mol. Gen. Genet. 116, 291 (1972). I°M. Friedkin and D. Roberts, J. Biol. Chem. 207, 245 (1954). 110. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265
(1951). lZT. B/icher, Biochim. Biophys. Acta 1,292 (1947).
[58]
THYM1DINE PHOSPHORYLASE
439
Purification of E n z y m e Is
Bacterial Strain. Best results are obtained with a strain of S. typhimurium which is constitutive for thymidine phosphorylase and defective in purine nucleoside phosphorylase. Although the following procedure can be used with wild-type strains (inducible for the enzyme and producing an active purine nucleoside phosphorylase), the final enzyme solution still contains trace amounts of purine nucleoside phosphorylase. A suitable strain (deoR c, deoD-) can be obtained by published procedures 7'9 or directly from the author.
Growth of Cells WILD-TYPE Salmonella typhimurium (ATCC 15277). Inoculate 2-liter flasks containing l-liter of Casamino acid medium, buffered at pH 7.0 with phosphate, with 25 ml of an overnight culture grown on the same medium. Incubate the flasks with shaking at 37 ° for 4 hr or until cells are in mid-log phase. Add sterile 2-deoxyribose to the cells to a final concentration of 0.2%. Continue incubation of the cells at 37 ° with aeration for an additional 90 min and then rapidly chill and harvest. Wash the cell paste in 1 mM EDTA and store frozen at -10 °.
Salmonella typhimurium
STRAINS CONSTITUTIVE
FOR THYMIDINE
PnOSPnORYLASE (deoRC). Inoculate 2-liter flasks containing l-liter of Casamino acid medium, buffered at pH 7.0 with phosphate and containing 10/zg/ml of thymine, with 25 ml of an overnight culture grown on the same medium. Incubate the flasks with shaking at 37 ° overnight and harvest in the morning. Wash the cell paste as above and store frozen at - 10°. The use of the constitutive strains is much more convenient since it does not require induction with 2-deoxyribose, and the amount of enzyme obtained is about 1.5-2 times the amount obtained from the same number of wild-type induced cells.
Preparation of Cell Extract. Resuspend 24 g of frozen S. typhimurium in 150 ml of 0.04 M triethanolamine buffer, pH 7.6, 1 mM in EDTA. Disrupt the cells in a Branson Sonifier keeping the mixture below 10° during oscillation with a NaCl-ice bath. Centrifuge the disrupted suspension for 45 rain at 27,000 g. Assay the supernatant fluid for activity and protein. Perform all subsequent procedures at 4 °.
Protamine Sulfate Step. Make a 2% solution of protamine sulfate (Eli Lilly). To each ml of extract add 0.05 ml of protamine sulfate, and after 10 min remove the precipitate by centrifugation. The enzyme activity 13Procedure based on work previously published: J. Blank and P. Hoffee, Arch. Biochem. Biophys. 168, 259 (1975).
440
PYRIMIDINE METABOLIZINGENZYMES
[581
should remain in the supernatant fluid. Add 0.2 ml of the 2% solution of protamine sulfate to each ml of the supernatant fluid. After 10 min collect the precipitate by centrifugation and dissolve it in 100 ml of 0.05 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Centrifuge the suspension and reserve the supernatant fluid for assay and further purification.
Ammonium Sulfate Fractionation. Bring the phosphate eluate to 40% ammonium sulfate saturation by the addition of 231 mg/ml of solid ammonium sulfate. After 30 min centrifuge the suspension and discard the precipitate. Bring the supernatant fluid to 60% ammonium sulfate saturation by the addition of 125 mg/ml of solid ammonium sulfate. After 30 min centrifuge the suspension. Dissolve the precipitate in 0.05 M potassium phosphate buffer, pH 7.5, and dialyze the solution overnight against 2000 volumes of 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. DEAE-Cellulose Chromatography. Apply the dialyzed ammonium sulfate fraction to a DEAE-cellulose column (2.5 x 27 cm) which has been equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Wash the column with about 200 ml of the equilibrating buffer or until no more 280 nm absorbing material is eluted. Thymidine phosphorylase is then eluted with a linear gradient of potassium phosphate buffer, from 0.01 M, pH 7.5 to 0.2 M, pH 8.0, 10 mM in 2-mercaptoethanol. Combine fractions containing enzyme activity and add sucrose to a final concentration of 15% (w/v). Bring the solution to 90% ammonium sulfate saturation by the slow addition of 630 mg/ml of solid ammonium sulfate. After 30 min centrifuge the suspension and dissolve the precipitate in 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Dialyze the solution overnight against 1000 ml of the same buffer. Sephadex G-150 Chromatography. Apply the dialyzed DEAE-cellulose fraction to a Sephadex G-150 column (2.5 x 70 cm) which has been equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Thymidine phosphorylase is eluted from the column by washing with the equilibration buffer. Fractions containing activity are pooled. Hydroxyapatite Chromatography. Apply the Sephadex G-150 fraction to a column (2.5 × 10 cm) of hydroxyapatite (Hypatite C, Clarkson Chemical Company, Inc., Williamsport, Pa.) which has been equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2mercaptoethanol. Thymidine phosphorylase does not adsorb to the
[58]
441
THYMIDINE PHOSPHORYLASE PURIFICATION PROCEDURE FOR THYMIDINE PHOSPHORYLASEFROM Salmonella
typhimurium a
Fraction 1. Crude extract 2. Protamine sulfate step 3. Ammonium sulfate (40-60% sat.) 4. DEAE-cellulose 5. Sephadex G-150 6. Hydroxyapatite
Volume (ml)
Total units
Specific Purification Recovery activityb (fold) (%)
133 106
5800 5500
1.8 5.8
1 3
100 94
9 16 32 2
4300 2500 1910 1180
17.4 45.5 200.0 465.0
10 25 111 258
74 43 33 20
a Data given for constitutive mutant JB-3041. Constitutive strains are available from the author or can be selected by procedures described in Blank and Hoffee? Specific activity is expressed in units/rag protein. One unit is defined as the number of /xmoles of thymine produced per minute at 37°. hydroxyapatite under these conditions and is eluted with the same buffer. Pool the fractions containing e n z y m e activity and add sucrose to a final concentration of 20%, Bring the solution to 90% ammonium sulfate saturation by the addition of 630 mg/ml of solid ammonium sulfate. After 30 min, centrifuge and dissolve the precipitate in 0.01 M potassium phosphate buffer, p H 7.5, 10 mM in 2-mercaptoethanol and 20% (w/v) in sucrose. This fraction is homogenous by polyacrylamide gel electrophoresis. The purification procedure is summarized in the table. P r o p e r t i e s la
Molecular Weight. Thymidine phosphorylase purified from S. typhimurium has a molecular weight o f 100,000 _ 10% and is c o m p o s e d of two subunits of molecular weight 47,000. Substrate Specificity. Purified thymidine phosphorylase can catalyze the conversion of thymidine and deoxyuridine to their respective bases and deoxyribose-l-phosphate. Both substrates show equal activity. Bromodeoxyuridine and iododeoxyuridine react at 40% the rate of thymidine. N o activity is seen with the following nucleosides: deoxycytidine, deoxyadenosine, deoxyguanosine, or uridine. The Michaelis constants for thymidine and deoxyuridine are 2.1 mM and 8.0 mM, respectively, and for phosphate and arsenate they are 2.3 mM and 1.3 M, respectively.
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
THYMIDINE PHOSPHORYLASE
437
at pH 7.4. Fraction 6 was used as enzyme; the measured Vmax with uridine as substrate was 63.5 /zmoles/min/mg of protein. The Km for Pi, determined with uridine (1.2 mM) as substrate, was 0.38 mM. Uridine concentrations above 15 mM were somewhat inhibitory; no other nucleoside substrate tested had this effect. The conclusion that a single pyrimidine nucleoside phosphorylase cleaves both uridine and thymidine in H. influenzae rests on several lines of evidence. First, the two activities maintained a constant ratio over a 400-fold purification and were not resolved by analytical polyacrylamide gel electrophoresis. Second, the activities showed identical kinetics of heat inactivation and were identically protected against heating by several nucleoside substrates. Third, kinetic studies with ribo- and deoxynucleoside substrate pairs gave results which conformed quantitatively with expectation for a single enzyme comprising both activities. 2 The pattern of substrate specificity observed is quite similar to that of the uridine phosphorylase purified from several mammalian sources, which were observed to retain significant activity toward deoxynucleosides. 5-g Bacillus stearothermophilus was found to possess a single pyrimidine nucleoside phosphorylase which was 1.5 times as active with thymidine as with uridinef this ratio for the H. influenzae enzyme was 0.25. The enzyme described here shows a striking preference for 5bromodeoxyuridine as a substrate; the initial rate observed with this substrate was 5 times that found with any other deoxynucleoside. ST. 6 E. 7A. 8 H. aP.
A. Krenitsky, M. Barclay, and J. A. Jacquez, J. Biol. Chem. 239, 805 (1964). W. Yamada, J. Biol. Chem. 243, 1649 (1968). Kraut and E. W. Yamada, J. Biol. Chem. 246, 2021 (1971). Pontis, G. Degerstedt, and P. Reichard, Biochim. Biophys. Acta 51, 139 (1961). P. Saunders, B. A. Wilson, and G. F. Saunders, J. Biol. Chem. 244, 3691 (1969).
[58] T h y m i d i n e
from Salmonella
Phosphorylase
typhim urium B y PATRICIA A . H O F F E E a n d JAMES B L A N K Thymidine + P~ ~ thymine + 2-deoxy-D-ribose-l-phosphate
In Escherichia coli and Salmonella typhimurium, thymidine phosphorylase plays an important role in the metabolism of thymine auxotrophs and is necessary for the conversion of exogenous thymine to thymidine. In low-thymine-requiring strains, thymidine phosphorylase becomes METHODS IN ENZYMOLOGY, VOL. LI
Copyright© 1978by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181951-5
438
PYRIMIDINE METABOLIZINGENZYMES
[58]
phenotypically constitutive in strains containing a mutation in deoxyribose-5-phosphate aldolase. 1-a Thymidine phosphorylase is regulated along with deoxyribose-5-phosphate aldolase, phosphodeoxyribomutase, and purine nucleoside phosphorylase as part of the deo regulon. 2-a
Assay Method 1° Principle. Enzyme activity is measured spectrophotometrically by measuring the formation of thymine from thymidine in the presence of sodium arsenate. Reagents Tris (hydroxymethyl) aminomethane-HC1 buffer 0.2 M, pH 7.4, with 0.1 M sodium arsenate Thymidine, 4 mg dissolved in 1 ml of the Tris.HC1 buffer NaOH 0.3 N Procedure. Add 0.2 ml of the thymidine-buffer solution to 3 tubes marked 0, 5, and 10 min. Add enzyme solution at 0°. To the 0 time tube add 1 ml of NaOH and incubate the 5- and 10-min tubes at 37°. Stop the reaction at the specified time by adding 1 ml of 0.3 N NaOH. The optical density of each sample is then determined in a spectrophotometer set at 300 nm. The increase in optical density is plotted against time. Definition o f Units. One unit of activity is defined as the number of /zmoles of thymine produced per minute at 37°. An extinction coefficient of 4.04 is used. Protein is determined by the method of Lowry et al. 11 before ammonium sulfate fractionation (see Purification) and by the method of B/icher 12after ammonium sulfate fractionation. 1W. E. Razzell and G. G. Khorana, Biochim. Biophys. Acta 28, 562 (1958). ~T. R. Breitman and R. M. Bradford, Biochim. Biophys. Acta 138, 217 (1967). aP. Hoffee, J. Bacteriol. 95, 449 (1968). 4A. Munch-Peterson. Fur. J. Biochem. 6, 432 (1968). ~M. S. Lomax and G. R. Greenberg. J. Bacteriol. 96, 501 (1968). 6p. Hoffee and B. C. Robertson, J. Bacteriol. 97, 1386 (1969). 7B. C. Robertson, P. Jargiello, J. Blank, and P. Hoffee, J. Baeteriol. 102, 628 (1970). s S. Ahmad and R. H. Pritchard, Mol. Gen. Genet. 104, 351 (1969). aJ. Blank and P. Hoffee, Mol. Gen. Genet. 116, 291 (1972). I°M. Friedkin and D. Roberts, J. Biol. Chem. 207, 245 (1954). 110. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265
(1951). lZT. B/icher, Biochim. Biophys. Acta 1,292 (1947).
[58]
THYM1DINE PHOSPHORYLASE
439
Purification of E n z y m e Is
Bacterial Strain. Best results are obtained with a strain of S. typhimurium which is constitutive for thymidine phosphorylase and defective in purine nucleoside phosphorylase. Although the following procedure can be used with wild-type strains (inducible for the enzyme and producing an active purine nucleoside phosphorylase), the final enzyme solution still contains trace amounts of purine nucleoside phosphorylase. A suitable strain (deoR c, deoD-) can be obtained by published procedures 7'9 or directly from the author.
Growth of Cells WILD-TYPE Salmonella typhimurium (ATCC 15277). Inoculate 2-liter flasks containing l-liter of Casamino acid medium, buffered at pH 7.0 with phosphate, with 25 ml of an overnight culture grown on the same medium. Incubate the flasks with shaking at 37 ° for 4 hr or until cells are in mid-log phase. Add sterile 2-deoxyribose to the cells to a final concentration of 0.2%. Continue incubation of the cells at 37 ° with aeration for an additional 90 min and then rapidly chill and harvest. Wash the cell paste in 1 mM EDTA and store frozen at -10 °.
Salmonella typhimurium
STRAINS CONSTITUTIVE
FOR THYMIDINE
PnOSPnORYLASE (deoRC). Inoculate 2-liter flasks containing l-liter of Casamino acid medium, buffered at pH 7.0 with phosphate and containing 10/zg/ml of thymine, with 25 ml of an overnight culture grown on the same medium. Incubate the flasks with shaking at 37 ° overnight and harvest in the morning. Wash the cell paste as above and store frozen at - 10°. The use of the constitutive strains is much more convenient since it does not require induction with 2-deoxyribose, and the amount of enzyme obtained is about 1.5-2 times the amount obtained from the same number of wild-type induced cells.
Preparation of Cell Extract. Resuspend 24 g of frozen S. typhimurium in 150 ml of 0.04 M triethanolamine buffer, pH 7.6, 1 mM in EDTA. Disrupt the cells in a Branson Sonifier keeping the mixture below 10° during oscillation with a NaCl-ice bath. Centrifuge the disrupted suspension for 45 rain at 27,000 g. Assay the supernatant fluid for activity and protein. Perform all subsequent procedures at 4 °.
Protamine Sulfate Step. Make a 2% solution of protamine sulfate (Eli Lilly). To each ml of extract add 0.05 ml of protamine sulfate, and after 10 min remove the precipitate by centrifugation. The enzyme activity 13Procedure based on work previously published: J. Blank and P. Hoffee, Arch. Biochem. Biophys. 168, 259 (1975).
440
PYRIMIDINE METABOLIZINGENZYMES
[581
should remain in the supernatant fluid. Add 0.2 ml of the 2% solution of protamine sulfate to each ml of the supernatant fluid. After 10 min collect the precipitate by centrifugation and dissolve it in 100 ml of 0.05 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Centrifuge the suspension and reserve the supernatant fluid for assay and further purification.
Ammonium Sulfate Fractionation. Bring the phosphate eluate to 40% ammonium sulfate saturation by the addition of 231 mg/ml of solid ammonium sulfate. After 30 min centrifuge the suspension and discard the precipitate. Bring the supernatant fluid to 60% ammonium sulfate saturation by the addition of 125 mg/ml of solid ammonium sulfate. After 30 min centrifuge the suspension. Dissolve the precipitate in 0.05 M potassium phosphate buffer, pH 7.5, and dialyze the solution overnight against 2000 volumes of 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. DEAE-Cellulose Chromatography. Apply the dialyzed ammonium sulfate fraction to a DEAE-cellulose column (2.5 x 27 cm) which has been equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Wash the column with about 200 ml of the equilibrating buffer or until no more 280 nm absorbing material is eluted. Thymidine phosphorylase is then eluted with a linear gradient of potassium phosphate buffer, from 0.01 M, pH 7.5 to 0.2 M, pH 8.0, 10 mM in 2-mercaptoethanol. Combine fractions containing enzyme activity and add sucrose to a final concentration of 15% (w/v). Bring the solution to 90% ammonium sulfate saturation by the slow addition of 630 mg/ml of solid ammonium sulfate. After 30 min centrifuge the suspension and dissolve the precipitate in 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Dialyze the solution overnight against 1000 ml of the same buffer. Sephadex G-150 Chromatography. Apply the dialyzed DEAE-cellulose fraction to a Sephadex G-150 column (2.5 x 70 cm) which has been equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2-mercaptoethanol. Thymidine phosphorylase is eluted from the column by washing with the equilibration buffer. Fractions containing activity are pooled. Hydroxyapatite Chromatography. Apply the Sephadex G-150 fraction to a column (2.5 × 10 cm) of hydroxyapatite (Hypatite C, Clarkson Chemical Company, Inc., Williamsport, Pa.) which has been equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, 10 mM in 2mercaptoethanol. Thymidine phosphorylase does not adsorb to the
[58]
441
THYMIDINE PHOSPHORYLASE PURIFICATION PROCEDURE FOR THYMIDINE PHOSPHORYLASEFROM Salmonella
typhimurium a
Fraction 1. Crude extract 2. Protamine sulfate step 3. Ammonium sulfate (40-60% sat.) 4. DEAE-cellulose 5. Sephadex G-150 6. Hydroxyapatite
Volume (ml)
Total units
Specific Purification Recovery activityb (fold) (%)
133 106
5800 5500
1.8 5.8
1 3
100 94
9 16 32 2
4300 2500 1910 1180
17.4 45.5 200.0 465.0
10 25 111 258
74 43 33 20
a Data given for constitutive mutant JB-3041. Constitutive strains are available from the author or can be selected by procedures described in Blank and Hoffee? Specific activity is expressed in units/rag protein. One unit is defined as the number of /xmoles of thymine produced per minute at 37°. hydroxyapatite under these conditions and is eluted with the same buffer. Pool the fractions containing e n z y m e activity and add sucrose to a final concentration of 20%, Bring the solution to 90% ammonium sulfate saturation by the addition of 630 mg/ml of solid ammonium sulfate. After 30 min, centrifuge and dissolve the precipitate in 0.01 M potassium phosphate buffer, p H 7.5, 10 mM in 2-mercaptoethanol and 20% (w/v) in sucrose. This fraction is homogenous by polyacrylamide gel electrophoresis. The purification procedure is summarized in the table. P r o p e r t i e s la
Molecular Weight. Thymidine phosphorylase purified from S. typhimurium has a molecular weight o f 100,000 _ 10% and is c o m p o s e d of two subunits of molecular weight 47,000. Substrate Specificity. Purified thymidine phosphorylase can catalyze the conversion of thymidine and deoxyuridine to their respective bases and deoxyribose-l-phosphate. Both substrates show equal activity. Bromodeoxyuridine and iododeoxyuridine react at 40% the rate of thymidine. N o activity is seen with the following nucleosides: deoxycytidine, deoxyadenosine, deoxyguanosine, or uridine. The Michaelis constants for thymidine and deoxyuridine are 2.1 mM and 8.0 mM, respectively, and for phosphate and arsenate they are 2.3 mM and 1.3 M, respectively.
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
