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NADPH-Dependent 15-hydroxyprostaglandin dehydrogenase and prostaglandin 9-ketoreductase from porcine kidney
142
ENZYMES AND RECEPTORS" PURIFICATION AND ASSAY
[23]
[23] P u r i f i c a t i o n o f N A D P + / N A D P H - D e p e n d e n t 15-Hydroxyprostaglandin Dehydrogenase and Prostaglandin 9-Ketoreductase from Porcine Kidney 1 B y H S I N - H S I U N G TAI a n d D A V I D G U E Y - B I N C H A N G
NADP+-dependent 15-hydroxyprostaglandin dehydrogenase (also known as type II 15-hydroxyprostaglandin dehydrogenase) was first known to catalyze the oxidation of 15(S)-hydroxyl group of prostaglandins to 15-ketoprostaglandins. ~ OH
OH
~ ' ~ • ' " ' ~ C O O H
• OH
+ NADP+ ~
~
~ - ] - ' " ' ~ ~
COOH
+ NADPH + H +
~ OH PGF2~
OH
O 15-keto-PGF2o
Subsequent studies showed that the same enzyme not only catalyzes reversible oxidoreduction of prostaglandins at C-15, but also affects reversible oxidoreduction of prostaglandins at C-9. 3"4These findings lead to the conclusion that the previously recognized NADP+-dependent 15-hydroxyprostaglandin dehydrogenase and prostaglandin 9-ketoreductase activities are in fact alternate activities of a single enzyme protein. The enzyme was shown to exist in multiple forms a-6 and was thought to control the PGE:PGF ratio and the duration of biologically active prostaglandins.7 The diversified functions of the enzyme reflect the versatility of the enzyme and the expression of each enzyme activity may depend on the availability of each particular prostaglandin substrate and the ratio of N A D P H : N A D P +. We and others have isolated two homogeneous forms of this enzyme from porcine kidney and human placenta, respectively. 3'4 This presentation describes our method of purification and some properties of the enzyme from porcine kidney. This work was supported in part by grants from the National Institutes of Health (GM25247) and the American Heart Association (78-865). 2 S. C. Lee and L. Levine, J. Biol. Chem. 250, 548 (1975). 3 y . M. Lin, and J. Jarabak, Biochem. Biophys. Res. Commun. 81, 1227 (1978). 4 D. G.-B. Chang and H. H. Tai, Biochem. Biophys. Res. Commun. 99, 745 (1981)• 5 S. C. Lee and L. Levine, J. Biol. Chem. 250, 4549 (1975). 6 A. Hassid and L. Levine, Prostaglandins 13, 503 (1977). T S. C. Lee, S. S. Pong, D. Katzen, K. Y. Wu, and L. Levine, Biochemistry 14, 142 (1975). 1
METHODS IN ENZYMOLOGY, VOL. 86
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181986-8
[23]
TYPE II 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE
143
Assay Method Principle. The assay of 15-hydroxyprostaglandin dehydrogenase activity can be done by a spectrophotometric method based on the conversion of PGE to 15-keto-PGE, which forms a chromophore (Em = 27,000 M -1 cm -1 at hmax = 500 rim) after alkalinization of the reaction mixture. 8 The activity can also be assayed spectrofluorometrically by following the increase in fluorescence of NADPH at 460 nm with excitation at 340 nm. Alternatively, the assay of 15-hydroxyprostaglandin dehydrogenase and prostaglandin 9-ketoreductase activities can be carried out by radioimmunological methods. The production of 15-keto-PGF2~ from PGF2~ (15-hydroxyprostaglandin dehydrogenase) or the formation of PGF2~ from PGE2 (prostaglandin 9-ketoreductase) can be determined quantitatively by specific radioimmunoassays for 15-keto-PGF2~ 9 or PGFz~ ,9 respectively. Reagents Potassium phosphate buffer, 0.1 M, pH 7.5 Tris-HC1, 0.05 M, pH 7.5, containing 0.1% gelatin (RIA buffer) NaOH, 2 N NADP + NADPH PGEz PGF2~
15-Keto-PGF2~ PGF2~ antisera9 15-Keto-PGF2~ antisera 9 PGF~-[lzsI]iodotyrosine methyl ester conjugate 1° 15-KetoPGFz~-[125I]iodotyrosine methyl ester conjugate 1° Charcoal suspension: 100 ml of RIA buffer containing 1 g of bovine 7-globulin and 3 g of Norit A) and enzyme Procedures Spectrophotometric or Spectrofluorometric Method. The assay mixture contains PGE2 (56.8 nmol), NADP + (200 nmol), and enzyme in a final volume of 1 ml of 0.1 M potassium phosphate, pH 7.5. The reaction is allowed to proceed at 37° for 40 min and then is terminated by the addition of 0.1 ml of 2 N NaOH. The transient chromophore generated is quantitated by measuring the absorption at 500 nm. If the enzyme activity is assayed spectrofluorometrically, the increase in fluorescence of NADPH at
8 E. Angg~rd,C. Larsson, and B. Samuelsson,Acta Physiol. Scand. 81, 396 (1971). 9 j. C. Cornette, K. L. Harrison, and K. T. Kirton, Prostaglandins $, 155 (1974). 10H. H. Tai and B. Yuan, Anal. Biochem. 87, 343 (1978).
144
ENZYMES AND RECEPTORS:
PURIFICATION
A N D ASSAY
[23]
460 nm with excitation at 340 nm is measured. The concentration of N A D P H is determined by assuming that Em = 6200 M -t cm -t at hmax = 340 nm. 11 Radioimmunological Method. The assay mixture contains PGE2 (14 nmol, for prostaglandin 9-ketoreductase) or PGF2~ (14 nmol, for 15-hydroxyprostaglandin dehydrogenase), N A D P H or NADP + (500 nmol), and enzyme in a final volume of 0.2 ml of 0.1 M potassium phosphate buffer, pH 7.5. The reaction is allowed to proceed at 37° for l0 min and then is terminated by boiling for 2 min. After removal of denatured proteins by centrifugation at 1000 g for 10 min, the supernatant is diluted and assayed for immunoreactive PGFz~ or 15-keto-PGFz,. The radioimmunoassay mixture contains 0.2 ml of standards or diluted sample, 0.1 ml of diluted PGF2~ or 15-keto-PGF2~ antisera, and 0.1 ml of the respective labeled hapten in 0.05 M Tris-HCl, pH 7.5, containing 0.1% gelatin. Each sample is run in duplicate. After 1 hr of incubation at room temperature, each tube receives 1 ml of HzO before addition of 0.2 ml of charcoal suspension and mixing in a vortex mixer. After standing for 5 min at room temperature, the mixture is centrifuged for 5 min at 1000 g. The supernatant containing the bound hapten is separated from the pellet containing the free hapten. The radioactivity in the supernatant and the pellet is separately determined by a gamma spectrometer. The ratio of cpm bound to the total cpm is calculated for each sample, and the concentration of each sample is determined from a standard curve after logit transformation as described by Tai and Chey. ~2 Purifiction of N A D P + / N A D P H - D e p e n d e n t 15-Hydroxyprostaglandin D e h y d r o g e n a s e and Prostaglandin 9-Ketoreductase from Porcine Kidney 4 Preparation o f Crude Enzyme Extract. All steps are performed at 4° . Porcine kidney (500 g) is homogenized in 1 liter of I0 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA (buffer A) in a Waring blender for 2 min. The homogenate is centrifuged at 40,000 g for 2 min. The supernatant is designated $4o. Acetone Fractionation. To $40 is added slowly prechilled acetone ( - 6 8 °) to 33% (v/v) with stirring for 10 min. The precipitate is removed by centrifugation at 40,000 g for 15 min. Acetone is further added to the supernatant to 43% with stirring for 10 min, and the precipitate is again removed by centrifugation at 40,000 g for 15 min. The supernatant is then brought to an acetone concentration of 67% and stirred for 10 min before
~ B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1949). ~2H. H. Tai and W. Y. Chey, Anal. Biochem. 74, 12 (1976).
[23]
TYPE II 15-HYDROXYPROSTAGLANDINDEHYDROGENASE
145
centrifugation at 40,000 g for 15 min. The precipitate is dissolved in 50 ml of buffer A and designated as the acetone fraction. Sephadex G-IO0 Chromatography. The acetone fraction is immediately applied to a Sephadex G-100 column (6 x 100 cm) equilibrated with buffer A. The column is eluted with the same buffer, and fractions of 13 ml are collected. The active fractions as determined by spectrophotometric method are pooled and concentrated to 20 ml using an Amicon ultrafiltration PM-10 membrane. TEAE-Cellulose Chromatography. The concentrated material is applied to a TEAE-cellulose column (1.5 x 25 cm) equilibrated with buffer A. The column is eluted with 10 mM potassium phosphate, pH 7.5, containing 1 mM EDTA (buffer B), and fractions of 13 ml are collected. The active fractions are pooled and concentrated to 20 ml by Amicon ultrafiltration. Isoelectric Focusing. The concentrated fraction from TEAE-cellulose chromatography is isoelectrofocused in a sucrose density gradient using 2.5% Ampholine (pH 4-6) in a 110-ml LKB column as described by Vesterberg et al.'3 Water at 3° is circulated to cool the column. Electrofocusing is performed at 1600 V for 16 hr. The contents of the column are collected in 1-ml fractions. Two peaks of activity appear and are designated form I and form II with pI values of 5.8 and 4.8, respectively. Active fractions from both peaks are concentrated separately and passed through a Sephadex G-50 (1 x 15 cm) column equilibrated and eluted with 50 mM potassium phosphate, pH 7.5, containing 1 mM EDTA. The active fractions are concentrated by lyophilization and dialyzed against buffer B for 24 hr. The enzyme is stored in aliquots at - 80°. A summary of the results of the purification of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) with its concurrent activities of prostaglandin 9-ketoreductase (9-PGKR), 9-hydroxyprostaglandin dehydrogenase (9-PGDH), and prostaglandin 15ketoreductase (15-PGKR) is shown in the table. Properties N
Homogeneity. The final preparations of both enzyme forms show single bands upon sodium dodecyl phosphate-polyacrylamide gel electrophoretic analysis. Molecular Weight. Both forms of the native enzyme contain a single polypeptide chain with a molecular weight of 29,000, although minor differences in amino acid composition are noted. ,30. Vesterberg, T. Wadstrom, K. Vesterberg, H. Svensson, and B. Malugren, Biochim. Biophys. Acta 133, 435 (1967). ~4 D. G.-B. Chang, and H. H. Tai, Arch. Biochern. Biophys. 214, 464 (1982).
146
ENZYMES A N D RECEPTORS: P U R I F I C A T I O N A N D ASSAY
i
e-,
-¥>~ ~
*"q "O
[h O ' -
m
z
"O e'~
>. <
u~
,-.Z
.=
e~
~~ M
O
~o~ ~,'o~ d,
e~
Z
e I
.~ "o
<
u ~>,,0
[23]
[24] NADP-LINKED PGD DEHYDROGENASE FROM SWINE BRAIN 147
Reversibility. The enzyme is able to catalyze reversible NADP÷/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 at pH 7.5. Substrate Specificity. For 15-hydroxyprostaglandin dehydrogenase activity, either form of the enzyme utilizes all kinds of prostaglandins as a substrate, PGB1 being the best substrate. For prostaglandin 9-ketoreductase activity, prostaglandins having 9-keto group can serve as substrate, PGA1-GSH being the best substrate. Km values for various prostaglandins have been determined.15 The enzyme does not catalyze oxidoreduction of prostaglandins at C-11. Coenzyme Specificity. N A D P + / N A D P H are preferred coenzyme for oxidoreduction of prostaglandins at C-9 and C-15. Km values for N A D W and N A D P H are 1.4 and 100/xM, respectively. Stability and Storage. The enzyme can be stored at - 8 0 ° for 1 year without appreciable loss of activity. 15 D. G.-B. Chang and H. H. Tai, Biochem. Biophys. Res. Commun. 101, 898 (1981).
[24] Isolation of NADP+-Dependent PGD,-Specific 15-Hydroxyprostaglandin Dehydrogenase from Swine Brain
By TAKAO SHIMIZU, KIKUKO WATANABE, HIDEKADO TOKUMOTO, and OSAMU HAYAISHI Prostaglandin D2 + NADP + ~ 15-ketoprostaglandin Da + NADPH + H ÷
The first step in the metabolic inactivation of prostaglandins is the oxidation of the 15-hydroxy group.l-4 Two types of 15-hydroxyprostaglandin dehydrogenase have been described (types I and 11).5-9 However, PGD2 was a poor substrate for these enzymes in terms of Vmax and Km values 2 ,l°-12 Ellis et al. 13 demonstrated that the major urinary metabolites of 1 E. ,~ngghrd and B. Samuelsson, J. Biol. Chem. 239, 4097 (1964). 2 E. ,~ngghrd and B. Samuelsson, Ark. Kemi 25, 293 (1966). 3 S. S. Braithwaite and J. Jarabak, J. Biol. Chem. 250, 2315 (1975). 4 j. Nakano, E. ,~ngg~trd, and B. Samuelsson, Eur. J. Biochem. 11, 386 (1969). 5 S.-C. Lee and L. Levine, J. Biol, Chem. 250, 548 (1975). 8 L. Kaplan and L. Levine, Arch. Biochem. Biophys. 167, 284 (1975). r S.-C. Lee, S.-S. Pong, D. Katzen, K.-Y. Wu, and L. Levine, Biochemistry 14, 142 (1975). a j. Jarabak and J. Fried, Prostaglandins 18, 241 (1979). a H. S. Hansen, Prostaglandins 12, 647 (1976). 10 F. F. Sun, S. B. Armour, V. R. Bockstanz, and J. C. McGuire, Adv. Prostaglandin Thromboxane Res. 1, 163 (1976).
METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, lnc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8
ENZYMES AND RECEPTORS" PURIFICATION AND ASSAY
[23]
[23] P u r i f i c a t i o n o f N A D P + / N A D P H - D e p e n d e n t 15-Hydroxyprostaglandin Dehydrogenase and Prostaglandin 9-Ketoreductase from Porcine Kidney 1 B y H S I N - H S I U N G TAI a n d D A V I D G U E Y - B I N C H A N G
NADP+-dependent 15-hydroxyprostaglandin dehydrogenase (also known as type II 15-hydroxyprostaglandin dehydrogenase) was first known to catalyze the oxidation of 15(S)-hydroxyl group of prostaglandins to 15-ketoprostaglandins. ~ OH
OH
~ ' ~ • ' " ' ~ C O O H
• OH
+ NADP+ ~
~
~ - ] - ' " ' ~ ~
COOH
+ NADPH + H +
~ OH PGF2~
OH
O 15-keto-PGF2o
Subsequent studies showed that the same enzyme not only catalyzes reversible oxidoreduction of prostaglandins at C-15, but also affects reversible oxidoreduction of prostaglandins at C-9. 3"4These findings lead to the conclusion that the previously recognized NADP+-dependent 15-hydroxyprostaglandin dehydrogenase and prostaglandin 9-ketoreductase activities are in fact alternate activities of a single enzyme protein. The enzyme was shown to exist in multiple forms a-6 and was thought to control the PGE:PGF ratio and the duration of biologically active prostaglandins.7 The diversified functions of the enzyme reflect the versatility of the enzyme and the expression of each enzyme activity may depend on the availability of each particular prostaglandin substrate and the ratio of N A D P H : N A D P +. We and others have isolated two homogeneous forms of this enzyme from porcine kidney and human placenta, respectively. 3'4 This presentation describes our method of purification and some properties of the enzyme from porcine kidney. This work was supported in part by grants from the National Institutes of Health (GM25247) and the American Heart Association (78-865). 2 S. C. Lee and L. Levine, J. Biol. Chem. 250, 548 (1975). 3 y . M. Lin, and J. Jarabak, Biochem. Biophys. Res. Commun. 81, 1227 (1978). 4 D. G.-B. Chang and H. H. Tai, Biochem. Biophys. Res. Commun. 99, 745 (1981)• 5 S. C. Lee and L. Levine, J. Biol. Chem. 250, 4549 (1975). 6 A. Hassid and L. Levine, Prostaglandins 13, 503 (1977). T S. C. Lee, S. S. Pong, D. Katzen, K. Y. Wu, and L. Levine, Biochemistry 14, 142 (1975). 1
METHODS IN ENZYMOLOGY, VOL. 86
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181986-8
[23]
TYPE II 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE
143
Assay Method Principle. The assay of 15-hydroxyprostaglandin dehydrogenase activity can be done by a spectrophotometric method based on the conversion of PGE to 15-keto-PGE, which forms a chromophore (Em = 27,000 M -1 cm -1 at hmax = 500 rim) after alkalinization of the reaction mixture. 8 The activity can also be assayed spectrofluorometrically by following the increase in fluorescence of NADPH at 460 nm with excitation at 340 nm. Alternatively, the assay of 15-hydroxyprostaglandin dehydrogenase and prostaglandin 9-ketoreductase activities can be carried out by radioimmunological methods. The production of 15-keto-PGF2~ from PGF2~ (15-hydroxyprostaglandin dehydrogenase) or the formation of PGF2~ from PGE2 (prostaglandin 9-ketoreductase) can be determined quantitatively by specific radioimmunoassays for 15-keto-PGF2~ 9 or PGFz~ ,9 respectively. Reagents Potassium phosphate buffer, 0.1 M, pH 7.5 Tris-HC1, 0.05 M, pH 7.5, containing 0.1% gelatin (RIA buffer) NaOH, 2 N NADP + NADPH PGEz PGF2~
15-Keto-PGF2~ PGF2~ antisera9 15-Keto-PGF2~ antisera 9 PGF~-[lzsI]iodotyrosine methyl ester conjugate 1° 15-KetoPGFz~-[125I]iodotyrosine methyl ester conjugate 1° Charcoal suspension: 100 ml of RIA buffer containing 1 g of bovine 7-globulin and 3 g of Norit A) and enzyme Procedures Spectrophotometric or Spectrofluorometric Method. The assay mixture contains PGE2 (56.8 nmol), NADP + (200 nmol), and enzyme in a final volume of 1 ml of 0.1 M potassium phosphate, pH 7.5. The reaction is allowed to proceed at 37° for 40 min and then is terminated by the addition of 0.1 ml of 2 N NaOH. The transient chromophore generated is quantitated by measuring the absorption at 500 nm. If the enzyme activity is assayed spectrofluorometrically, the increase in fluorescence of NADPH at
8 E. Angg~rd,C. Larsson, and B. Samuelsson,Acta Physiol. Scand. 81, 396 (1971). 9 j. C. Cornette, K. L. Harrison, and K. T. Kirton, Prostaglandins $, 155 (1974). 10H. H. Tai and B. Yuan, Anal. Biochem. 87, 343 (1978).
144
ENZYMES AND RECEPTORS:
PURIFICATION
A N D ASSAY
[23]
460 nm with excitation at 340 nm is measured. The concentration of N A D P H is determined by assuming that Em = 6200 M -t cm -t at hmax = 340 nm. 11 Radioimmunological Method. The assay mixture contains PGE2 (14 nmol, for prostaglandin 9-ketoreductase) or PGF2~ (14 nmol, for 15-hydroxyprostaglandin dehydrogenase), N A D P H or NADP + (500 nmol), and enzyme in a final volume of 0.2 ml of 0.1 M potassium phosphate buffer, pH 7.5. The reaction is allowed to proceed at 37° for l0 min and then is terminated by boiling for 2 min. After removal of denatured proteins by centrifugation at 1000 g for 10 min, the supernatant is diluted and assayed for immunoreactive PGFz~ or 15-keto-PGFz,. The radioimmunoassay mixture contains 0.2 ml of standards or diluted sample, 0.1 ml of diluted PGF2~ or 15-keto-PGF2~ antisera, and 0.1 ml of the respective labeled hapten in 0.05 M Tris-HCl, pH 7.5, containing 0.1% gelatin. Each sample is run in duplicate. After 1 hr of incubation at room temperature, each tube receives 1 ml of HzO before addition of 0.2 ml of charcoal suspension and mixing in a vortex mixer. After standing for 5 min at room temperature, the mixture is centrifuged for 5 min at 1000 g. The supernatant containing the bound hapten is separated from the pellet containing the free hapten. The radioactivity in the supernatant and the pellet is separately determined by a gamma spectrometer. The ratio of cpm bound to the total cpm is calculated for each sample, and the concentration of each sample is determined from a standard curve after logit transformation as described by Tai and Chey. ~2 Purifiction of N A D P + / N A D P H - D e p e n d e n t 15-Hydroxyprostaglandin D e h y d r o g e n a s e and Prostaglandin 9-Ketoreductase from Porcine Kidney 4 Preparation o f Crude Enzyme Extract. All steps are performed at 4° . Porcine kidney (500 g) is homogenized in 1 liter of I0 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA (buffer A) in a Waring blender for 2 min. The homogenate is centrifuged at 40,000 g for 2 min. The supernatant is designated $4o. Acetone Fractionation. To $40 is added slowly prechilled acetone ( - 6 8 °) to 33% (v/v) with stirring for 10 min. The precipitate is removed by centrifugation at 40,000 g for 15 min. Acetone is further added to the supernatant to 43% with stirring for 10 min, and the precipitate is again removed by centrifugation at 40,000 g for 15 min. The supernatant is then brought to an acetone concentration of 67% and stirred for 10 min before
~ B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1949). ~2H. H. Tai and W. Y. Chey, Anal. Biochem. 74, 12 (1976).
[23]
TYPE II 15-HYDROXYPROSTAGLANDINDEHYDROGENASE
145
centrifugation at 40,000 g for 15 min. The precipitate is dissolved in 50 ml of buffer A and designated as the acetone fraction. Sephadex G-IO0 Chromatography. The acetone fraction is immediately applied to a Sephadex G-100 column (6 x 100 cm) equilibrated with buffer A. The column is eluted with the same buffer, and fractions of 13 ml are collected. The active fractions as determined by spectrophotometric method are pooled and concentrated to 20 ml using an Amicon ultrafiltration PM-10 membrane. TEAE-Cellulose Chromatography. The concentrated material is applied to a TEAE-cellulose column (1.5 x 25 cm) equilibrated with buffer A. The column is eluted with 10 mM potassium phosphate, pH 7.5, containing 1 mM EDTA (buffer B), and fractions of 13 ml are collected. The active fractions are pooled and concentrated to 20 ml by Amicon ultrafiltration. Isoelectric Focusing. The concentrated fraction from TEAE-cellulose chromatography is isoelectrofocused in a sucrose density gradient using 2.5% Ampholine (pH 4-6) in a 110-ml LKB column as described by Vesterberg et al.'3 Water at 3° is circulated to cool the column. Electrofocusing is performed at 1600 V for 16 hr. The contents of the column are collected in 1-ml fractions. Two peaks of activity appear and are designated form I and form II with pI values of 5.8 and 4.8, respectively. Active fractions from both peaks are concentrated separately and passed through a Sephadex G-50 (1 x 15 cm) column equilibrated and eluted with 50 mM potassium phosphate, pH 7.5, containing 1 mM EDTA. The active fractions are concentrated by lyophilization and dialyzed against buffer B for 24 hr. The enzyme is stored in aliquots at - 80°. A summary of the results of the purification of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) with its concurrent activities of prostaglandin 9-ketoreductase (9-PGKR), 9-hydroxyprostaglandin dehydrogenase (9-PGDH), and prostaglandin 15ketoreductase (15-PGKR) is shown in the table. Properties N
Homogeneity. The final preparations of both enzyme forms show single bands upon sodium dodecyl phosphate-polyacrylamide gel electrophoretic analysis. Molecular Weight. Both forms of the native enzyme contain a single polypeptide chain with a molecular weight of 29,000, although minor differences in amino acid composition are noted. ,30. Vesterberg, T. Wadstrom, K. Vesterberg, H. Svensson, and B. Malugren, Biochim. Biophys. Acta 133, 435 (1967). ~4 D. G.-B. Chang, and H. H. Tai, Arch. Biochern. Biophys. 214, 464 (1982).
146
ENZYMES A N D RECEPTORS: P U R I F I C A T I O N A N D ASSAY
i
e-,
-¥>~ ~
*"q "O
[h O ' -
m
z
"O e'~
>. <
u~
,-.Z
.=
e~
~~ M
O
~o~ ~,'o~ d,
e~
Z
e I
.~ "o
<
u ~>,,0
[23]
[24] NADP-LINKED PGD DEHYDROGENASE FROM SWINE BRAIN 147
Reversibility. The enzyme is able to catalyze reversible NADP÷/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 at pH 7.5. Substrate Specificity. For 15-hydroxyprostaglandin dehydrogenase activity, either form of the enzyme utilizes all kinds of prostaglandins as a substrate, PGB1 being the best substrate. For prostaglandin 9-ketoreductase activity, prostaglandins having 9-keto group can serve as substrate, PGA1-GSH being the best substrate. Km values for various prostaglandins have been determined.15 The enzyme does not catalyze oxidoreduction of prostaglandins at C-11. Coenzyme Specificity. N A D P + / N A D P H are preferred coenzyme for oxidoreduction of prostaglandins at C-9 and C-15. Km values for N A D W and N A D P H are 1.4 and 100/xM, respectively. Stability and Storage. The enzyme can be stored at - 8 0 ° for 1 year without appreciable loss of activity. 15 D. G.-B. Chang and H. H. Tai, Biochem. Biophys. Res. Commun. 101, 898 (1981).
[24] Isolation of NADP+-Dependent PGD,-Specific 15-Hydroxyprostaglandin Dehydrogenase from Swine Brain
By TAKAO SHIMIZU, KIKUKO WATANABE, HIDEKADO TOKUMOTO, and OSAMU HAYAISHI Prostaglandin D2 + NADP + ~ 15-ketoprostaglandin Da + NADPH + H ÷
The first step in the metabolic inactivation of prostaglandins is the oxidation of the 15-hydroxy group.l-4 Two types of 15-hydroxyprostaglandin dehydrogenase have been described (types I and 11).5-9 However, PGD2 was a poor substrate for these enzymes in terms of Vmax and Km values 2 ,l°-12 Ellis et al. 13 demonstrated that the major urinary metabolites of 1 E. ,~ngghrd and B. Samuelsson, J. Biol. Chem. 239, 4097 (1964). 2 E. ,~ngghrd and B. Samuelsson, Ark. Kemi 25, 293 (1966). 3 S. S. Braithwaite and J. Jarabak, J. Biol. Chem. 250, 2315 (1975). 4 j. Nakano, E. ,~ngg~trd, and B. Samuelsson, Eur. J. Biochem. 11, 386 (1969). 5 S.-C. Lee and L. Levine, J. Biol, Chem. 250, 548 (1975). 8 L. Kaplan and L. Levine, Arch. Biochem. Biophys. 167, 284 (1975). r S.-C. Lee, S.-S. Pong, D. Katzen, K.-Y. Wu, and L. Levine, Biochemistry 14, 142 (1975). a j. Jarabak and J. Fried, Prostaglandins 18, 241 (1979). a H. S. Hansen, Prostaglandins 12, 647 (1976). 10 F. F. Sun, S. B. Armour, V. R. Bockstanz, and J. C. McGuire, Adv. Prostaglandin Thromboxane Res. 1, 163 (1976).
METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, lnc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8