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[47] d -galactitol-6-phosphate dehydrogenase
[47]
D-GALACTITOL-6-PHOSPHATE DEHYDROGENASE
275
trate, malate, phosphoenolpyruvate, or p y r u v a t e , N A D P H and N A D H inhibit the e n z y m e completely at a final concentration of 1 m M when either N A D or N A D P is used as electron acceptor. F u r t h e r m o r e , 1 m M ATP causes an e n z y m e inhibition of 35% with N A D and 16% with N A D P as electron acceptor.
[47] By
D-Galactitol-6-phosphate
1 Dehydrogenase
R I C H A R D L . A N D E R S O N a n d J O H N P. M A R K W E L L
D-Galactitol 6-phosphate 1 + NAD+---~ D-tagatose 6-phosphate + N A D H + H +
This inducible e n z y m e functions in the catabolism of galactitol in Klebsiella pneumoniae. 2,a
Assay Method 3 Principle. The rate of D-galactitol 6-phosphate-dependent N A D H formation is determined b y measuring the rate of a b s o r b a n c e increase at 340 nm. Reagents Tris-HC1 buffer, 0.2 M, p H 8.5 N A D +, 50 m M D-Galactitol 6-phosphate, 4 22.5 m M MnCI2, 2 m M Procedure. The following components are added to a microcuvette with a 1.0-cm light path: 50 ~1 of buffer, 20 /zl of N A D +, 20 /zl of D-galactitol 6-phosphate, 5/.d of MnC12, e n z y m e , and w a t e r to a volume of 0.15 ml. The reaction is initiated b y the addition of D-galactitol'6phosphate dehydrogenase. The reaction rates are conveniently m e a s u r e d with a Gilford multiple-sample absorbance-recording s p e c t r o p h o t o m e t e r . The cuvette c o m p a r t m e n t should be thermostatted at 3ft. Definition o f Unit and Specific Activity. One unit of D-galactitol-6phosphate dehydrogenase is defined as the a m o u n t that catalyzes the 1 D-Galactitol 6-phosphate is s o m e t i m e s referred to as L-galactitol 1-phosphate, w hi c h is the
same compound. 2 j. Markwell, G. T. Shimamoto, D. L. Bissett, and R. L. Anderson, Biochem. Biophys. Res. Commun. 71, 221 (1976). 3 j. p. Markwell and R. L. Anderson, Arch. Biochem. Biophys. 209, 592 (1981). 4 j. B. Wolff and N. O. Kaplan, J. Biol. Chem. 218, 849 (1956).
METHODS IN ENZYMOLOGY,VOL. 89
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181989-2
276
OXIDATION-REDUCTION ENZYMES
[47]
PURIFICATION OF D-GALACTITOL-6-PHOSPHATE DEHYDROGENASE
Fraction
Volume (ml)
Cell extract DEAE-cellulose Ammonium sulfate Sephadex G-150
21.8 28.0 2.2 7.7
Protein (mg) 296 35.0 16.3 2.4
Activity (units) 15.9 11.8 9.85 3.26
Specific activity (units/mg protein) 0.053 0.34 0.61 1.4
Recovery (%) 100 74 62 20
reduction of 1 /zmol of NA-D+ per minute in the standard assay. Specific activity (units per milligram of protein) is based on protein determinations by the Lowry procedure. Alternative Assay Procedure. The reverse reaction may also be measured spectrophotometrically. 3
Purification Procedure 3
Organism and Growth Conditions. The bacterial strain used is Klebsiella pneumoniae PRL-R3 (formerly designated Aerobacter aerogenes PRLR3). It is grown aerobically at 30° in l-liter volumes of a medium containing 0.15% KH2PO4, 0.71% Na2HPO4, 0.3% (NH4)2SO4, 0.01% MgSO4, 0.0005% FeSO4 • 7 H~O, and 0.5% galactitol (autoclaved separately). The cells are harvested by centrifugation and washed with 0.85% (w/v) NaC1. Preparation of Cell Extracts. Pelleted cells are suspended in buffer A [0.025 M Tris-HCl (pH 8.5), 10% (v/v) glycerol, and 0.5 mM dithiothreitol] and broken by sonic treatment (10,000 Hz) in a Raytheon Model DF-101 (250-W) sonic oscillator cooled with circulating ice water. The broken-cell suspension is centrifuged at 12,000 g for 10 min, and the resulting supernatant fluid is designated the cell extract. General. The following procedures are performed at 0-4 °. A summary of the purification is shown in the table. DEAE-Cellulose Chromatography. The cell extract is applied to a column (1.2 × 6 cm) of DEAE-cellulose that has been equilibrated with buffer A. Protein is then eluted with a 200-ml linear gradient of 0 to 0.35 M KC1 in buffer A. Fractions (1.8 ml) are collected, and those that contain the peak of the activity (fractions 28 through 43) are pooled. Ammonium Sulfate Fractionation. To the above pooled fractions, ammonium sulfate (192 g/liter) is added slowly with stirring. After 15 min the resulting suspension is clarified by centrifugation and a second portion of
[47]
D-GALACTITOL-6-PHOSPHATE DEHYDROGENASE
277
ammonium sulfate (220 g/liter) is added. After 15 min, the precipitate is collected by centrifugation and dissolved in 2 ml of buffer A. Sephadex G-150 Chromatography. The above fraction is chromatographed on a column (1.4 x 82 cm) of Sephadex G-150 equilibrated with buffer A. The protein is eluted with the same buffer. Fractions (1.3 ml) are collected, and those that contain the peak of the activity (fractions 55 through 60) are pooled. The enzyme is 26-fold purified with a 20% over-all recovery. Properties 3
Substrate Specificity. D-Galactitol 6-phosphate (Km = 0.2 mM) and D-tagatose 6-phosphate (Kin = 0.1 mM) are the only known substrates. Compounds (10 raM) that did not serve as substrates (<0.1% of the rate with D-galactitol 6-phosphate) in the presence of NAD + were D-mannitol 6-phosphate, D-glucitol 6-phosphate, D-glucose 6-phosphate, D-galactose 6-phosphate, and D-fructose 1-phosphate. Compounds (10 raM) that did not serve as substrates (<0.1% of the rate with D-tagatose 6-phosphate) in the presence of NADH were D-fructose 6-phosphate, D-fructose 1-phosphate, D-fructose 1,6-bisphosphate, D-glucose 6-phosphate, D-galactose 6-phosphate, and L-sorbose 1-phosphate. No activity was detected when NADP + and NADPH were substituted for NAD + and NADH, respectively, with any of the sugar phosphates tested. Effect of Divalent Metal Ions. A divalent metal is required both for activity and stability. Treatment of the enzyme with l0 mM EDTA followed by removal of this chelating agent by passage of the enzyme through a column of Sephadex G-25 absolished all activity unless a divalent metal ion was included in the assay mixture. If the metal salt (67 t~M) was added within 15 rain of the EDTA treatment, the following restorations (%) of activity were achieved: CdSO4, 91; MnCl2, 71; CoC12, 61; ZnC12, 51. If the metal-ion addition was delayed for 150 min, only 4% of the activity could be restored. pH Optimum. Activity as a function of pH is maximal at about pH 8.7 in Tris-HC1 buffer. Molecular Weight. An estimation of the molecular weight by Sephadex G-150 chromatography yielded a value of 85,000. Stability. D-Galactitol-6-phosphate dehydrogenase in the Sephadex G-150 fractions retained most of its activity for at least 2 weeks when stored at 0° or - 2 0 °.
D-GALACTITOL-6-PHOSPHATE DEHYDROGENASE
275
trate, malate, phosphoenolpyruvate, or p y r u v a t e , N A D P H and N A D H inhibit the e n z y m e completely at a final concentration of 1 m M when either N A D or N A D P is used as electron acceptor. F u r t h e r m o r e , 1 m M ATP causes an e n z y m e inhibition of 35% with N A D and 16% with N A D P as electron acceptor.
[47] By
D-Galactitol-6-phosphate
1 Dehydrogenase
R I C H A R D L . A N D E R S O N a n d J O H N P. M A R K W E L L
D-Galactitol 6-phosphate 1 + NAD+---~ D-tagatose 6-phosphate + N A D H + H +
This inducible e n z y m e functions in the catabolism of galactitol in Klebsiella pneumoniae. 2,a
Assay Method 3 Principle. The rate of D-galactitol 6-phosphate-dependent N A D H formation is determined b y measuring the rate of a b s o r b a n c e increase at 340 nm. Reagents Tris-HC1 buffer, 0.2 M, p H 8.5 N A D +, 50 m M D-Galactitol 6-phosphate, 4 22.5 m M MnCI2, 2 m M Procedure. The following components are added to a microcuvette with a 1.0-cm light path: 50 ~1 of buffer, 20 /zl of N A D +, 20 /zl of D-galactitol 6-phosphate, 5/.d of MnC12, e n z y m e , and w a t e r to a volume of 0.15 ml. The reaction is initiated b y the addition of D-galactitol'6phosphate dehydrogenase. The reaction rates are conveniently m e a s u r e d with a Gilford multiple-sample absorbance-recording s p e c t r o p h o t o m e t e r . The cuvette c o m p a r t m e n t should be thermostatted at 3ft. Definition o f Unit and Specific Activity. One unit of D-galactitol-6phosphate dehydrogenase is defined as the a m o u n t that catalyzes the 1 D-Galactitol 6-phosphate is s o m e t i m e s referred to as L-galactitol 1-phosphate, w hi c h is the
same compound. 2 j. Markwell, G. T. Shimamoto, D. L. Bissett, and R. L. Anderson, Biochem. Biophys. Res. Commun. 71, 221 (1976). 3 j. p. Markwell and R. L. Anderson, Arch. Biochem. Biophys. 209, 592 (1981). 4 j. B. Wolff and N. O. Kaplan, J. Biol. Chem. 218, 849 (1956).
METHODS IN ENZYMOLOGY,VOL. 89
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181989-2
276
OXIDATION-REDUCTION ENZYMES
[47]
PURIFICATION OF D-GALACTITOL-6-PHOSPHATE DEHYDROGENASE
Fraction
Volume (ml)
Cell extract DEAE-cellulose Ammonium sulfate Sephadex G-150
21.8 28.0 2.2 7.7
Protein (mg) 296 35.0 16.3 2.4
Activity (units) 15.9 11.8 9.85 3.26
Specific activity (units/mg protein) 0.053 0.34 0.61 1.4
Recovery (%) 100 74 62 20
reduction of 1 /zmol of NA-D+ per minute in the standard assay. Specific activity (units per milligram of protein) is based on protein determinations by the Lowry procedure. Alternative Assay Procedure. The reverse reaction may also be measured spectrophotometrically. 3
Purification Procedure 3
Organism and Growth Conditions. The bacterial strain used is Klebsiella pneumoniae PRL-R3 (formerly designated Aerobacter aerogenes PRLR3). It is grown aerobically at 30° in l-liter volumes of a medium containing 0.15% KH2PO4, 0.71% Na2HPO4, 0.3% (NH4)2SO4, 0.01% MgSO4, 0.0005% FeSO4 • 7 H~O, and 0.5% galactitol (autoclaved separately). The cells are harvested by centrifugation and washed with 0.85% (w/v) NaC1. Preparation of Cell Extracts. Pelleted cells are suspended in buffer A [0.025 M Tris-HCl (pH 8.5), 10% (v/v) glycerol, and 0.5 mM dithiothreitol] and broken by sonic treatment (10,000 Hz) in a Raytheon Model DF-101 (250-W) sonic oscillator cooled with circulating ice water. The broken-cell suspension is centrifuged at 12,000 g for 10 min, and the resulting supernatant fluid is designated the cell extract. General. The following procedures are performed at 0-4 °. A summary of the purification is shown in the table. DEAE-Cellulose Chromatography. The cell extract is applied to a column (1.2 × 6 cm) of DEAE-cellulose that has been equilibrated with buffer A. Protein is then eluted with a 200-ml linear gradient of 0 to 0.35 M KC1 in buffer A. Fractions (1.8 ml) are collected, and those that contain the peak of the activity (fractions 28 through 43) are pooled. Ammonium Sulfate Fractionation. To the above pooled fractions, ammonium sulfate (192 g/liter) is added slowly with stirring. After 15 min the resulting suspension is clarified by centrifugation and a second portion of
[47]
D-GALACTITOL-6-PHOSPHATE DEHYDROGENASE
277
ammonium sulfate (220 g/liter) is added. After 15 min, the precipitate is collected by centrifugation and dissolved in 2 ml of buffer A. Sephadex G-150 Chromatography. The above fraction is chromatographed on a column (1.4 x 82 cm) of Sephadex G-150 equilibrated with buffer A. The protein is eluted with the same buffer. Fractions (1.3 ml) are collected, and those that contain the peak of the activity (fractions 55 through 60) are pooled. The enzyme is 26-fold purified with a 20% over-all recovery. Properties 3
Substrate Specificity. D-Galactitol 6-phosphate (Km = 0.2 mM) and D-tagatose 6-phosphate (Kin = 0.1 mM) are the only known substrates. Compounds (10 raM) that did not serve as substrates (<0.1% of the rate with D-galactitol 6-phosphate) in the presence of NAD + were D-mannitol 6-phosphate, D-glucitol 6-phosphate, D-glucose 6-phosphate, D-galactose 6-phosphate, and D-fructose 1-phosphate. Compounds (10 raM) that did not serve as substrates (<0.1% of the rate with D-tagatose 6-phosphate) in the presence of NADH were D-fructose 6-phosphate, D-fructose 1-phosphate, D-fructose 1,6-bisphosphate, D-glucose 6-phosphate, D-galactose 6-phosphate, and L-sorbose 1-phosphate. No activity was detected when NADP + and NADPH were substituted for NAD + and NADH, respectively, with any of the sugar phosphates tested. Effect of Divalent Metal Ions. A divalent metal is required both for activity and stability. Treatment of the enzyme with l0 mM EDTA followed by removal of this chelating agent by passage of the enzyme through a column of Sephadex G-25 absolished all activity unless a divalent metal ion was included in the assay mixture. If the metal salt (67 t~M) was added within 15 rain of the EDTA treatment, the following restorations (%) of activity were achieved: CdSO4, 91; MnCl2, 71; CoC12, 61; ZnC12, 51. If the metal-ion addition was delayed for 150 min, only 4% of the activity could be restored. pH Optimum. Activity as a function of pH is maximal at about pH 8.7 in Tris-HC1 buffer. Molecular Weight. An estimation of the molecular weight by Sephadex G-150 chromatography yielded a value of 85,000. Stability. D-Galactitol-6-phosphate dehydrogenase in the Sephadex G-150 fractions retained most of its activity for at least 2 weeks when stored at 0° or - 2 0 °.