538
[73]
PURINE M E T A B O L I Z I N G ENZYMES
Activation of enzymic activity is observed at high concentrations of nucleoside substrates. After electrophoretic fractionation of the enzyme, it is seen that activation is more pronounced in the acidic variants than in the basic variants, m'za Treatment with 5,5'-dithiobis(2-nitrobenzoic acid) results in a 60% decrease in activity and loss of substrate activation. Both the activity and the phenomenon of substrate activation are fully recovered after dithiothreitol treatment.2s Sulfhydryl groups play a key role in the enzymic behavior. With labeled PCMB it is possible to titrate 12 -SH groups per mole of enzyme. Activity is lost after the first 3-4 -SH are reacted but may be fully restored with dithiothreitol. As noted above, 5,5'-dithiobis(2-nitrobenzoic acid) reacts with only 2-4 sulfhydryl groups causing partial loss of activity; however, in the presence of SDS, approximately 12 -SH groups are titratable, zs The effect of pH on kinetic parameters suggests an essential role for cysteine and histidine in catalysis. 2 At pH values below 6 and above 8, substrate activation and inhibition, respectively, are observed with inorganic phosphate, z The synthetic reaction is favored with a Keq value of 54 for both inosine and deoxyinosine at pH 7.4, 24°.29'a° 2sR. P. Agarwaland R. E. Parks, Jr., J. Biol. Chem. 246, 3763 (1971). 2~H. M. Kalckar,J. Biol. Chem. 167, 477 (1947). 3 0 M. Friedkin,J. Biol. Chem. 184, 449 (1950).
[73] C h i n e s e
Hamster
Purine Nucleoside
Phosphorylase 1
B y GREGORY MILMAN ',synthesis"
(Hypoxanthineor guanine) + ribose-l-P .
• (inosineor guanosine) + Pi
"breakdown"
Assay Method Principle. Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) in eukaryotes catalyzes the reversible conversion between the purine bases, hypoxanthine and guanine, and their corresponding nucleosides, inosine and guanosine. NUCLEOSIDE SYNTHESIS ASSAY. The formation of inosine or guanosine is measured in a radioisotope assay in which 14C-labeled purine base
1Abbreviations used are: P and Pi, phosphate and inorganic phosphate, respectively; Tricine, N-Tris(hydroxmethyl)methylglycine. METHODS IN ENZYMOLOGY,VOL. LI
Copyright © 1978 by Academic Press, Inc.
Allrightsofreproducfoninanyformreserved. ISBN (k12-181951-5
[73]
HAMSTER PURINE NUCLEOSIDE PHOSPHORYLASE
539
is converted to labeled nucleoside. In 1 N NH4OH, the labeled nucleoside passes through Cu r+ Chelex while the labeled purine base is bound. NUCLEOSIDE PHOSPHORYLASEASSAY. The conversion of nucleoside to purine base and ribose 1-phosphate is measured by the formation of [14C]ribose 1-phosphate from U-~4C-labeled nucleoside. In water, the unreacted nucleoside and purine base produced during the enzyme reaction bind to Cu r+ Chelex, while the [14C]ribose 1-P is eluted.
Reagents NH4OH, 1 N and 5 N Cu r÷ Chelex columns: Typically, 250 g of Chelex 100 (Bio-Rad) are allowed to stand overnight in 2 liters of 0.5 M CuCI2, and then are washed with 10 liters of HzO and 1 liter of 1 N NH4OH and stored in 1 N NH4OH. This preparation is used for the nucleoside synthesis assay. For the nucleoside phosphorylase assay, the Cu 2+ Chelex is rinsed 10 times, each time with 2 liters of H~O and stored in H20. Approximately 1-1.5 ml Cu r+ Chelex is placed into Pasteur pipet columns (3.5 × 0.5 cm) containing a glass-fiber plug. We usually use Cu r+ Chelex columns only once. However, they can be regenerated, first by washing with 25 ml of 5 N NH4OH, and then with either 12 ml of 1 N NH4OH for the nucleoside synthesis assay or with 25 ml of H20 for the nucleoside phosphorylase assay. A convenient reservoir is a plastic syringe attached to the top of the column with a short piece of rubber tubing. Enzyme buffer: Used to dilute enzyme samples, it consists of 20 mM Tris.HC1 (pH 7.8), 6 mM MgCI2, 100 mM KC1, 0.1 mM EDTA, and 0.5 mM dithiotreitol. Assay buffer: Consists of 500 mM Tris'HCl (pH 7.8), 50 mM MgCI~, 20 mM dithiotreitol Ribose l-phosphate, 11.5 mM Guanine (or hypoxanthine), 0.8 mM Guanosine (or inosine), 0.8 mM Potassium phosphate (pH 7.8), 100 mM [8-14C]guanine (or [8-14C]hypoxanthine), specific activity approximately 50 mCi/mmole. Background cpm in the absence of enzyme of less than 0.5% of the initial cpm in the assay are obtained by purifying the 14C-labeled purine bases from the suppliers prior to use. 14C-labeled purine base is absorbed to a standard Cu +2 Chelex column in 1 N NH4OH, washed with 5 ml 1 N NH4OH, eluted with 5 N NH4OH, and lyophilized. [U-~4C]guanosine (or [U-14C]inosine), specific activity approximately 200-500 mCi/mmole.
540
PURINE METABOLIZING ENZYMES
[73]
Nucleoside synthesis assay reagent: An assay reagent for 500 assays is prepared by combining 5 ml assay buffer, 5 ml ribose 1phosphate, 5 ml guanine (or hypoxanthine), approximately 5 × 107 cpm of [8-14C]guanine (or hypoxanthine), and H~O in a final volume of 20 ml. Nucleoside phosphorylase assay reagent: An assay reagent for 500 assays is prepared by combining 5 ml assay buffer, 5 ml potassium phosphate, 5 ml guanosine (or inosine), approximately 2 × 107 cpm of [U-14C]guanosine (or inosine), and H20 in a final volume of 1 ml. The assay reagents are frozen in 1-ml portions (25 assays) and are stable for months in a liquid nitrogen freezer. Scintillation fluid: Consists of 9.1 g of 2-5-diphenyloxazole (PPO), 0.61 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene (POPOP), 2140 ml of toluene, and 1250 ml of Triton X-100.
Procedure NUCLEOSIDE SYNTHESIS ASSAY. A reaction is initiated by adding 40 /~1 of nucleoside synthesis assay reagent to 60 ttl of enzyme sample in enzyme buffer in a 10 × 75 mm test tube on ice, mixing for approximately 1 sec on a Vortex mixer, and placing the reaction mixture in a 37 ° water bath. In some cases 10/zl of 1 mg/ml bovine serum albumin are included in the assay to improve the stability of the more purified enzyme samples. After 15 min, the incubation is terminated by adding 150 gl of 1 N NH4OH, mixing well, and placing the mixture on ice. A 200-/xl sample of each reaction is applied to a Cu ~+ Chelex column equilibrated in 1 N NH4OH. Nucleoside is eluted with 1.5 ml of 1 N NH4OH, collected directly into a scintillation vial, and counted in 10 ml of scintillation fluid. NUCLEOSIDE PHOSPHORYLASEASSAY. A reaction is initiated by adding 40/zl of nucleoside phosphorylase assay reagent to 60/zl of enzyme sample in enzyme buffer. After 15 min, the incubation is terminated by adding 150 /xl of HzO and immediately placing the mixture in an 80 ° water bath for 10 min. A 200-/xl sample of each cooled reaction mixture is applied to a Cu 2÷ Chelex column equilibrated in H~O. Ribose 1phosphate is eluted with 1.5 ml of HzO, collected directly into scintillation vials, and counted in 10 ml of scintillation fluid.
Definition of Unit and Specific Activity. A unit is defined as the amount of enzyme which catalyzed the formation of 1 gmole of guanosine per minute at 37 ° in the nucleoside synthesis assay. The number of nanomoles of substrate in a standard assay is determined from the amount of unlabeled and radioisotope-labeled purine base. The specific activity of the purine base is determined from the number of
[73]
HAMSTER PURINE NUCLEOSIDE PHOSPHORYLASE
541
cpm in an assay counted in the same manner as the assay samples. The nanomoles of product formed in the reaction is 1.25 times the product eluted from the Cu 2÷ Chelex columns. Specific activity is expressed as units of enzyme per milligram of protein determined by the method of Lowry et al. 2 with crystalline bovine serum albumin as a standard. Purification Procedure Purification from Chinese Hamster V79 Tissue Culture Cells. All procedures are performed at 0-5 ° unless otherwise specified. All enzyme fractions are stored in a liquid-nitrogen freezer. Enzyme activity is measured by the formation of guanosine from guanine and ribose 1phosphate in the nucleoside synthesis assay described above. A summary of a sample purification is presented in the table. Crude Extract. Chinese hamster V79 cells z are grown in suspension to a density of 0.5-1 x 106/ml in Joklik's minimum essential media' supplemented with nonessential amino acids, 1 mM sodium pyruvate, 50 mM Tricine (pH 7.6), and 5% fetal bovine serum. The cells are harvested by centrifugation and resuspended in 2 volumes of enzyme buffer (assay reagent). The cells are lysed by freezing and stored at - 7 0 °. The lysate from 25 ml of packed cells is thawed and centrifuged at 20,000 g for 20 min, and the supernatant fluid is saved. The pellet is resuspended in an additional 10 ml of the same buffer and centrifuged as PURIFICATION OF CHINESE HAMSTER V79 TISSUE CULTURE CELL PURINE NUCLEOSIDE PHOSPHORYLASE
Fraction Crude extract High-speed supernatant DEAE-Sephadex G-100 Sephadex Isoelectric focusing
Volume (ml)
Protein (mg)
76 73 2.0 1.4 6.0
1626 920 39 5.3 0.5 b
Specific Cumulative activity recovery Purification (units/rag) (%) (fold) 0.14 0.16 1.59 11.8~' 60.0 a
100 65 27 27 13
1 1.1 11 84 430
Assay includes 0.1 mg/ml of bovine serum albumin. Protein determined from intensity of enzyme band on sodium dodecyl sulfatepolyacrylamide gel in comparison with protein standards. 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). ZD. K. Ford and G. Yerganion, J. Natl. Cancer Inst. 21, (1958).
542
PURINE METABOLIZINGENZYMES
[73]
described above. The two supernatant fluids are combined to form the crude extract.
High-Speed Supernatant Fraction. The crude extract is centrifuged for 2.5 hr at 60,000 g in a Spinco 40 rotor. The supernatant fluid forms the high-speed supernatant fraction. DEAE-Sephadex Fraction. The high-speed supernatant fraction is applied to a column (0.9 x 20 cm) containing DEAE-Sephadex A-50 equilibrated in enzyme buffer. The column is washed with 40 ml of the same buffer, and then the enzyme is eluted in a 400-ml linear KCI gradient (100-250 mM) in enzyme buffer. Fractions of 4 ml are collected and assayed for enzyme activity which elutes in an irregular peak from 140-180 mM KC1. Only the most active fractions containing peak enzyme activity, approximately 80 ml, are pooled and concentrated to about 2 ml using an Amicon ultra.filtration cell with a PM-10 membrane. G-IO0 Sephadex Fraction. DEAE-Sephadex fraction enzyme is applied to a column (1.5 x 90 cm) containing G-100 Sephadex equilibrated in enzyme buffer. The column is developed with the same buffer, and 2ml fractions are collected and assayed for enzyme activity. Fractions containing enzyme activity (approximately 16 ml) are pooled and concentrated to about 1.5 ml using an Amicon ultrafiltration cell with a PM10 membrane. Isoelectric Focusing Fraction. A 110-ml, 0--46% linear sucrose gradient with 1% pH 5-7 ampholytes and 2 mM dithiothreitol is prepared manually in an LKB Model 8180 ampholine column following the procedure in the LKB manual. G-100 Sephadex fraction enzyme is applied to the middle of the gradient replacing the "light" solution in that region. Electrofocusing is conducted for 3-5 days at 3-5 °. During that time, the voltage is increased from 300 to 800 V. One-milliliter fractions are collected, and the pH and enzyme activity are measured. Fractions containing peak enzyme activity (2-3 fractions) are pooled. Properties 4
Chinese hamster pudne nucleoside phosphorylase from both V79 tissue culture cells and Chinese hamster organs appears to have identical structural and catalytic properties. At 37 ° the purified enzyme converts 60/.~moles of guanine to guanosine per minute per milligram of protein. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels indicates 4Reprinted with permission from G. Milman, D. L. Anton, and J. L. Weber, Biochemistry 15, 4967 (1976). Copyright by the American Chemical Society.
[74]
HYPOXANTHINE PHOSPHORIBOSY LTRANSFERASE
543
that the enzyme is composed of identical subunits of 30,000 molecular weight. The native enzyme behaves as a mixture of dimers of 68,000 molecular weight and trimers of 89,000 molecular weight during Sephadex G-100 chromatography. Sucrose gradient centrifugation indicates that the enzyme has a sedimentation coefficient of 5.4 S, which corresponds to a molecular weight of 94,000 and suggests a trimer structure. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants of 20 ~ for both hypoxanthine and guanine, 35/~M for guanosine, 50/zM for inosine, and 200/zM for both ribose 1-P and Pi. During electrofocusing, the enzyme forms a single major band at a pI of 5.25.
[74] Hypoxanthine Phosphoribosyltransferase from Chinese Hamster Brain and H u m a n Erythrocytes I By ANNE S. OLSEN and GREGORY MILMAN Hypoxanthine or guanine + PRPP
M#÷
--, IMP or GMP + PP,
Assay Method
Principle. Hypo×anthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is a salvage enzyme responsible for the conversion of the purine bases hypoxanthine or guanine to the corresponding 5'-ribonucleotides IMP or GMP. The formation of IMP or GMP is measured in a radioisotope assay in which 14C-labeled purine base is converted to the labeled nucleotide. The uncharged purine base is separated from the negatively charged nucleotide on DEAE-cellulose. Reagents Enzyme buffer, used to dilute enzyme samples, consisting of 20 mM Tris'HC1 (pH 7.8), 20 mM KC1, 6 mM MgClz, 0.1 mM EDTA, and 0.5 mM dithiothreitol Assay buffer, consisting of 500 mM Tris.HC1 (pH 7.8), 5 mM MgC12, and 20 mM dithiothreitol PRPP (sodium salt), 10 mM Hypoxanthine (or guanine), 0.6 mM; add HC1 to dissolve guanine 1The abbreviations used are: PRPP, 5-phosphoribosyl 1-pyrophosphate; PP~, inorganic pyrophosphate, HPRT, hypoxanthine phosphoribosyltransferase. METHODS
IN
ENZYMOLOGY,
VOL. LI
Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
[73]
PURINE M E T A B O L I Z I N G ENZYMES
Activation of enzymic activity is observed at high concentrations of nucleoside substrates. After electrophoretic fractionation of the enzyme, it is seen that activation is more pronounced in the acidic variants than in the basic variants, m'za Treatment with 5,5'-dithiobis(2-nitrobenzoic acid) results in a 60% decrease in activity and loss of substrate activation. Both the activity and the phenomenon of substrate activation are fully recovered after dithiothreitol treatment.2s Sulfhydryl groups play a key role in the enzymic behavior. With labeled PCMB it is possible to titrate 12 -SH groups per mole of enzyme. Activity is lost after the first 3-4 -SH are reacted but may be fully restored with dithiothreitol. As noted above, 5,5'-dithiobis(2-nitrobenzoic acid) reacts with only 2-4 sulfhydryl groups causing partial loss of activity; however, in the presence of SDS, approximately 12 -SH groups are titratable, zs The effect of pH on kinetic parameters suggests an essential role for cysteine and histidine in catalysis. 2 At pH values below 6 and above 8, substrate activation and inhibition, respectively, are observed with inorganic phosphate, z The synthetic reaction is favored with a Keq value of 54 for both inosine and deoxyinosine at pH 7.4, 24°.29'a° 2sR. P. Agarwaland R. E. Parks, Jr., J. Biol. Chem. 246, 3763 (1971). 2~H. M. Kalckar,J. Biol. Chem. 167, 477 (1947). 3 0 M. Friedkin,J. Biol. Chem. 184, 449 (1950).
[73] C h i n e s e
Hamster
Purine Nucleoside
Phosphorylase 1
B y GREGORY MILMAN ',synthesis"
(Hypoxanthineor guanine) + ribose-l-P .
• (inosineor guanosine) + Pi
"breakdown"
Assay Method Principle. Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) in eukaryotes catalyzes the reversible conversion between the purine bases, hypoxanthine and guanine, and their corresponding nucleosides, inosine and guanosine. NUCLEOSIDE SYNTHESIS ASSAY. The formation of inosine or guanosine is measured in a radioisotope assay in which 14C-labeled purine base
1Abbreviations used are: P and Pi, phosphate and inorganic phosphate, respectively; Tricine, N-Tris(hydroxmethyl)methylglycine. METHODS IN ENZYMOLOGY,VOL. LI
Copyright © 1978 by Academic Press, Inc.
Allrightsofreproducfoninanyformreserved. ISBN (k12-181951-5
[73]
HAMSTER PURINE NUCLEOSIDE PHOSPHORYLASE
539
is converted to labeled nucleoside. In 1 N NH4OH, the labeled nucleoside passes through Cu r+ Chelex while the labeled purine base is bound. NUCLEOSIDE PHOSPHORYLASEASSAY. The conversion of nucleoside to purine base and ribose 1-phosphate is measured by the formation of [14C]ribose 1-phosphate from U-~4C-labeled nucleoside. In water, the unreacted nucleoside and purine base produced during the enzyme reaction bind to Cu r+ Chelex, while the [14C]ribose 1-P is eluted.
Reagents NH4OH, 1 N and 5 N Cu r÷ Chelex columns: Typically, 250 g of Chelex 100 (Bio-Rad) are allowed to stand overnight in 2 liters of 0.5 M CuCI2, and then are washed with 10 liters of HzO and 1 liter of 1 N NH4OH and stored in 1 N NH4OH. This preparation is used for the nucleoside synthesis assay. For the nucleoside phosphorylase assay, the Cu 2+ Chelex is rinsed 10 times, each time with 2 liters of H~O and stored in H20. Approximately 1-1.5 ml Cu r+ Chelex is placed into Pasteur pipet columns (3.5 × 0.5 cm) containing a glass-fiber plug. We usually use Cu r+ Chelex columns only once. However, they can be regenerated, first by washing with 25 ml of 5 N NH4OH, and then with either 12 ml of 1 N NH4OH for the nucleoside synthesis assay or with 25 ml of H20 for the nucleoside phosphorylase assay. A convenient reservoir is a plastic syringe attached to the top of the column with a short piece of rubber tubing. Enzyme buffer: Used to dilute enzyme samples, it consists of 20 mM Tris.HC1 (pH 7.8), 6 mM MgCI2, 100 mM KC1, 0.1 mM EDTA, and 0.5 mM dithiotreitol. Assay buffer: Consists of 500 mM Tris'HCl (pH 7.8), 50 mM MgCI~, 20 mM dithiotreitol Ribose l-phosphate, 11.5 mM Guanine (or hypoxanthine), 0.8 mM Guanosine (or inosine), 0.8 mM Potassium phosphate (pH 7.8), 100 mM [8-14C]guanine (or [8-14C]hypoxanthine), specific activity approximately 50 mCi/mmole. Background cpm in the absence of enzyme of less than 0.5% of the initial cpm in the assay are obtained by purifying the 14C-labeled purine bases from the suppliers prior to use. 14C-labeled purine base is absorbed to a standard Cu +2 Chelex column in 1 N NH4OH, washed with 5 ml 1 N NH4OH, eluted with 5 N NH4OH, and lyophilized. [U-~4C]guanosine (or [U-14C]inosine), specific activity approximately 200-500 mCi/mmole.
540
PURINE METABOLIZING ENZYMES
[73]
Nucleoside synthesis assay reagent: An assay reagent for 500 assays is prepared by combining 5 ml assay buffer, 5 ml ribose 1phosphate, 5 ml guanine (or hypoxanthine), approximately 5 × 107 cpm of [8-14C]guanine (or hypoxanthine), and H~O in a final volume of 20 ml. Nucleoside phosphorylase assay reagent: An assay reagent for 500 assays is prepared by combining 5 ml assay buffer, 5 ml potassium phosphate, 5 ml guanosine (or inosine), approximately 2 × 107 cpm of [U-14C]guanosine (or inosine), and H20 in a final volume of 1 ml. The assay reagents are frozen in 1-ml portions (25 assays) and are stable for months in a liquid nitrogen freezer. Scintillation fluid: Consists of 9.1 g of 2-5-diphenyloxazole (PPO), 0.61 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene (POPOP), 2140 ml of toluene, and 1250 ml of Triton X-100.
Procedure NUCLEOSIDE SYNTHESIS ASSAY. A reaction is initiated by adding 40 /~1 of nucleoside synthesis assay reagent to 60 ttl of enzyme sample in enzyme buffer in a 10 × 75 mm test tube on ice, mixing for approximately 1 sec on a Vortex mixer, and placing the reaction mixture in a 37 ° water bath. In some cases 10/zl of 1 mg/ml bovine serum albumin are included in the assay to improve the stability of the more purified enzyme samples. After 15 min, the incubation is terminated by adding 150 gl of 1 N NH4OH, mixing well, and placing the mixture on ice. A 200-/xl sample of each reaction is applied to a Cu ~+ Chelex column equilibrated in 1 N NH4OH. Nucleoside is eluted with 1.5 ml of 1 N NH4OH, collected directly into a scintillation vial, and counted in 10 ml of scintillation fluid. NUCLEOSIDE PHOSPHORYLASEASSAY. A reaction is initiated by adding 40/zl of nucleoside phosphorylase assay reagent to 60/zl of enzyme sample in enzyme buffer. After 15 min, the incubation is terminated by adding 150 /xl of HzO and immediately placing the mixture in an 80 ° water bath for 10 min. A 200-/xl sample of each cooled reaction mixture is applied to a Cu 2÷ Chelex column equilibrated in H~O. Ribose 1phosphate is eluted with 1.5 ml of HzO, collected directly into scintillation vials, and counted in 10 ml of scintillation fluid.
Definition of Unit and Specific Activity. A unit is defined as the amount of enzyme which catalyzed the formation of 1 gmole of guanosine per minute at 37 ° in the nucleoside synthesis assay. The number of nanomoles of substrate in a standard assay is determined from the amount of unlabeled and radioisotope-labeled purine base. The specific activity of the purine base is determined from the number of
[73]
HAMSTER PURINE NUCLEOSIDE PHOSPHORYLASE
541
cpm in an assay counted in the same manner as the assay samples. The nanomoles of product formed in the reaction is 1.25 times the product eluted from the Cu 2÷ Chelex columns. Specific activity is expressed as units of enzyme per milligram of protein determined by the method of Lowry et al. 2 with crystalline bovine serum albumin as a standard. Purification Procedure Purification from Chinese Hamster V79 Tissue Culture Cells. All procedures are performed at 0-5 ° unless otherwise specified. All enzyme fractions are stored in a liquid-nitrogen freezer. Enzyme activity is measured by the formation of guanosine from guanine and ribose 1phosphate in the nucleoside synthesis assay described above. A summary of a sample purification is presented in the table. Crude Extract. Chinese hamster V79 cells z are grown in suspension to a density of 0.5-1 x 106/ml in Joklik's minimum essential media' supplemented with nonessential amino acids, 1 mM sodium pyruvate, 50 mM Tricine (pH 7.6), and 5% fetal bovine serum. The cells are harvested by centrifugation and resuspended in 2 volumes of enzyme buffer (assay reagent). The cells are lysed by freezing and stored at - 7 0 °. The lysate from 25 ml of packed cells is thawed and centrifuged at 20,000 g for 20 min, and the supernatant fluid is saved. The pellet is resuspended in an additional 10 ml of the same buffer and centrifuged as PURIFICATION OF CHINESE HAMSTER V79 TISSUE CULTURE CELL PURINE NUCLEOSIDE PHOSPHORYLASE
Fraction Crude extract High-speed supernatant DEAE-Sephadex G-100 Sephadex Isoelectric focusing
Volume (ml)
Protein (mg)
76 73 2.0 1.4 6.0
1626 920 39 5.3 0.5 b
Specific Cumulative activity recovery Purification (units/rag) (%) (fold) 0.14 0.16 1.59 11.8~' 60.0 a
100 65 27 27 13
1 1.1 11 84 430
Assay includes 0.1 mg/ml of bovine serum albumin. Protein determined from intensity of enzyme band on sodium dodecyl sulfatepolyacrylamide gel in comparison with protein standards. 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). ZD. K. Ford and G. Yerganion, J. Natl. Cancer Inst. 21, (1958).
542
PURINE METABOLIZINGENZYMES
[73]
described above. The two supernatant fluids are combined to form the crude extract.
High-Speed Supernatant Fraction. The crude extract is centrifuged for 2.5 hr at 60,000 g in a Spinco 40 rotor. The supernatant fluid forms the high-speed supernatant fraction. DEAE-Sephadex Fraction. The high-speed supernatant fraction is applied to a column (0.9 x 20 cm) containing DEAE-Sephadex A-50 equilibrated in enzyme buffer. The column is washed with 40 ml of the same buffer, and then the enzyme is eluted in a 400-ml linear KCI gradient (100-250 mM) in enzyme buffer. Fractions of 4 ml are collected and assayed for enzyme activity which elutes in an irregular peak from 140-180 mM KC1. Only the most active fractions containing peak enzyme activity, approximately 80 ml, are pooled and concentrated to about 2 ml using an Amicon ultra.filtration cell with a PM-10 membrane. G-IO0 Sephadex Fraction. DEAE-Sephadex fraction enzyme is applied to a column (1.5 x 90 cm) containing G-100 Sephadex equilibrated in enzyme buffer. The column is developed with the same buffer, and 2ml fractions are collected and assayed for enzyme activity. Fractions containing enzyme activity (approximately 16 ml) are pooled and concentrated to about 1.5 ml using an Amicon ultrafiltration cell with a PM10 membrane. Isoelectric Focusing Fraction. A 110-ml, 0--46% linear sucrose gradient with 1% pH 5-7 ampholytes and 2 mM dithiothreitol is prepared manually in an LKB Model 8180 ampholine column following the procedure in the LKB manual. G-100 Sephadex fraction enzyme is applied to the middle of the gradient replacing the "light" solution in that region. Electrofocusing is conducted for 3-5 days at 3-5 °. During that time, the voltage is increased from 300 to 800 V. One-milliliter fractions are collected, and the pH and enzyme activity are measured. Fractions containing peak enzyme activity (2-3 fractions) are pooled. Properties 4
Chinese hamster pudne nucleoside phosphorylase from both V79 tissue culture cells and Chinese hamster organs appears to have identical structural and catalytic properties. At 37 ° the purified enzyme converts 60/.~moles of guanine to guanosine per minute per milligram of protein. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels indicates 4Reprinted with permission from G. Milman, D. L. Anton, and J. L. Weber, Biochemistry 15, 4967 (1976). Copyright by the American Chemical Society.
[74]
HYPOXANTHINE PHOSPHORIBOSY LTRANSFERASE
543
that the enzyme is composed of identical subunits of 30,000 molecular weight. The native enzyme behaves as a mixture of dimers of 68,000 molecular weight and trimers of 89,000 molecular weight during Sephadex G-100 chromatography. Sucrose gradient centrifugation indicates that the enzyme has a sedimentation coefficient of 5.4 S, which corresponds to a molecular weight of 94,000 and suggests a trimer structure. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants of 20 ~ for both hypoxanthine and guanine, 35/~M for guanosine, 50/zM for inosine, and 200/zM for both ribose 1-P and Pi. During electrofocusing, the enzyme forms a single major band at a pI of 5.25.
[74] Hypoxanthine Phosphoribosyltransferase from Chinese Hamster Brain and H u m a n Erythrocytes I By ANNE S. OLSEN and GREGORY MILMAN Hypoxanthine or guanine + PRPP
M#÷
--, IMP or GMP + PP,
Assay Method
Principle. Hypo×anthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is a salvage enzyme responsible for the conversion of the purine bases hypoxanthine or guanine to the corresponding 5'-ribonucleotides IMP or GMP. The formation of IMP or GMP is measured in a radioisotope assay in which 14C-labeled purine base is converted to the labeled nucleotide. The uncharged purine base is separated from the negatively charged nucleotide on DEAE-cellulose. Reagents Enzyme buffer, used to dilute enzyme samples, consisting of 20 mM Tris'HC1 (pH 7.8), 20 mM KC1, 6 mM MgClz, 0.1 mM EDTA, and 0.5 mM dithiothreitol Assay buffer, consisting of 500 mM Tris.HC1 (pH 7.8), 5 mM MgC12, and 20 mM dithiothreitol PRPP (sodium salt), 10 mM Hypoxanthine (or guanine), 0.6 mM; add HC1 to dissolve guanine 1The abbreviations used are: PRPP, 5-phosphoribosyl 1-pyrophosphate; PP~, inorganic pyrophosphate, HPRT, hypoxanthine phosphoribosyltransferase. METHODS
IN
ENZYMOLOGY,
VOL. LI
Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5
