C Y T O C H R O MP' E450LTBAND INACTIVATION OF
[35]
LTB4
319
5-1ipoxygenase may undergo a product-mediated oxidative activation step; however, the chemical mechanism of such a step remains to be elucidated. As previously mentioned, 5-1ipoxygenase undergoes firstorder irreversible inactivation during the course of its reaction. 4'5 Interestingly, a similar enzyme inactivation occurs upon the exposure of the enzyme to fatty acid hydroperoxide in the absence of substrate turnover. It is possible, therefore, that the product-activated form of the enzyme is unstable. Studies have been performed on the effects of various stimulatory factors on human 5-1ipoxygenase kinetics. 5 These have shown that ATP, the 100,000 g pellet, and the 60-90% precipitate all stimulate the initial velocity of the reaction, and that the increased total product synthesis induced by none of these factors is due to a decreased rate of enzyme inactivation. Interestingly, the 60-90% precipitate actually increases the enzyme inactivation rate as well as the initial velocity. The effects of ATP are not influenced by temperature, but those of both the 100,000 g pellet and the 60-90% precipitate are augmented with increasing temperature. Much remains to be determined concerning the regulation and activation of 5-1ipoxygenase in the intact leukocyte. However, recent evidence indicating that a Ca2÷-dependent association of the enzyme with membrane occurs in ionophore-challenged leukocytes suggests possible roles for both Ca 2+ and membrane in the regulation of 5-1ipoxygenase activity. ~0,~8 Acknowledgments The authors acknowledge the contributions of many friends and colleagues at the Karolinska Institute and the Merck Frosst Centre for Therapeutic Research. We also gratefully acknowledge the support of the Swedish Medical Research Council (Grant # 03X-217), and we thank Ms. Barbara Pearce for excellent secretarial services. t8 C. A. Rouzer and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 84, 7933 (1987).
[35] C y t o c h r o m e P-450LTI3 a n d I n a c t i v a t i o n o f Leukotriene B 4
By RoY J. SOBERMAN Introduction
5(S),12(R)-Dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid [leukotriene B4 (LTB4)] is generally considered the most potent chemotactic compound yet described for human polymorphonuclear leukoMETHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990 by Academic Press, Inc. All fightsof reproductionin any formreserved.
320
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[35]
cytes (PMN). The biological functions of chemotaxis and receptor binding of LTB4 are inactivated by progressive oxidation of C:20.1-4 The o~-oxidation of this pathway is initiated by the enzymatic action of P'450LTB, a cytochrome P-450 mixed-function oxidase located, apparently exclusively, in human PMN. 5-1° Cytochrome P'450LTB is distinct from the oJ-hydroxylases which o~-oxidize prostaglandins and lauric acid by both its substrate specificity9 and lack of immunological crossreactivity with polyclonal antibody to both these enzymes. 9 Cytochrome P-450LTB shows a highly restricted substrate specificity, requiring the "correct" chirality of both the 5- and 12-hydroxyl group configuration and the cis and trans configuration of the double bonds and hydroxyl groups for maximal substrate activity. Cytochrome P-450LTB catalyzes the following reactions: LTB4 + Oz + NADPH + H + ~ 20-OH-LTB4 + NADP ÷ + HzO H 20-OH-LTB4 - (R-C-OH) + NADPH + 02
I
n
(1)
~ 20-CHO (20-aldehyde) - LTB4
o
II (R-C-H) + NADP ÷ + H20
(2)
The overall reaction catalyzed by this enzyme is LTB4 + 2NADPH + 202 ~ 20-CHO-LTB4 + 2NAD + + 2H20
The second reaction, the apparent direct enzymatic oxidation of 20OH-LTB4 by a cytochrome P-450, actually results from the enzymatic addition of a second hydroxyl group followed by the spontaneous dehydration of a dihydroxylated intermediate to an aldehyde. 20-CHO-LTB4 can also be hydroxylated to 20-COOH-LTB4 by cytochrome P'450LTB, but this reaction is relatively slow. l G. Hansson, J. ~. Lindgren, S.-E. Dahl6n, P. Hedqvist, and B. Samuelsson, FEBS Lett. 130, 107 (1981). 2 W. Jubiz, O. Rhdmark, C. Malmsten, G. Hansson, J./~. Lindgren, J. P~lmblad, A.-M. Ud~n, and B. Samuelsson, J. Biol. Chem. 257, 6106 (1982). 3 S. Shak and I. M. Goldstein, J. Biol. Chem. 259, 10181 (1984). 4 W. S. Powell, J. Biol. Chem. 259, 3082 (1984). H. Sumimoto, K. Takeshige, H. Sakai, and S. Minakami, Biochem. Biophys. Res. Commun. 125, 615 (1984). 6 S. Shak and I. M. Goldstein, Biochem. Biophys. Res. Commun. 123, 475 (1984). 7 S. Shak and I. M. Goldstein, J. Clin. Invest. 76, 1218 (1985). 8 R. J. Soberman, T. W. Harper, R. C. Murphy, and K. F. Austen, Proc. Natl. Acad. Sci. U.S.A. 82, 2292 (1985). 9 R. J. Soberman, R. T. Okita, B. Fitzsimmons, J. Rokach, B. Spur, and K. F. Austen, J. Biol. Chem. 262, 12421 (1987). lo R. J. Soberman, J. P. Sutyak, R. T. Okita, D. F. Wendelborn, L. J. Roberts II, and K. F. Austen, J. Biol. Chem. 2.63, 7996 (1988).
[35]
C Y T O C H R O MP-450LTB E AND INACTIVATION OF
LTB4
321
Principle. The conversion of LTB4 to 20-OH-LTB4 and the conversion of 20-OH-LTB4 to 20-CHO-LTB4 and 20-COOH-LTB4 is measured by reversed-phase high-performance liquid chromatography (RP-HPLC) at 280 nm and quantitated by integrated absorbance. Procedures
Reagents. LTB4 and 20-OH LTB4 can be purchased from either Cayman Chemical Company Corporation (Ann Arbor, MI) or Biomol Corporation (Philadelphia, PA). NADP and NADPH are available from Sigma (St. Louis, MO). Methanol HPLC (grade) is obtained from Burdick and Jackson. Acetic acid (reagent grade) and ammonium acetate (HPLC grade) are purchased from Fischer Chemical Corporation. PMN and PMN microsomes are prepared exactly as described by Soberman et al. 9'1° and in this series. ~ Stock Solutions Reaction buffer, 50 mM Tris-HC1, pH 8.0 NADPH, 1.0 mM in Buffer A Substrate, 150/~M LTB4 or 20-OH-LTB4 in ethanol PMN microsomes, generally 0.5-1.0 mg/ml
Preparation of 20-CHO LTB4 Approximately 500 units of equine alcohol dehydrogenase are diluted to 200 ml with 50 mM Tris-HCl buffer (pH 7.0) and dialyzed against 6 liters of this buffer for 48 h to remove the ethanol present in the commercial enzyme preparation. 20-OH-LTB4 (100 lzg) in methanol is then evaporated to dryness under nitrogen in a 250-ml beaker and then resuspended in 80 ml of 0.2 M glycine buffer (pH 10.0; the optimal buffer for alcohol dehydrogenase). NAD + is then added to a concentration of 500/zM, and 54 units of the dialyzed alcohol dehydrogenase added to initiate the reaction. The reaction is allowed to proceed for 90 min at room temperature and then terminated by the addition of 100 ml of stop solution. Twenty 10-ml aliquots are removed, diluted to a volume of 50 ml with cold (4°) distilled water (pH 5.6), and separately concentrated by application onto a single reversed-phase Sep-Pak column (Waters Associates, Milford, MA) previously prepared by washing with 20 ml of methanol, followed by 20 ml of distilled water. The Sep-Pak column is then washed with 20 ml of water, adjusted to pH 5.6 with 0.6 N NaOH, and substrate and products eluted with 2 ml of methanol. The methanol eluate fraction is evaporated to a H R. J. Soberman and R. T. Okita, this series, Vol. 163, p. 349.
322
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[35]
volume of 0.75 ml under nitrogen, mixed with an equal volume of 50 mM ammonium acetate (pH 5.6), and injected onto a 5-/~m reversed-phase HPLC column (0.46 x 25 cm) equilibrated in a solvent of methanol/50 mM ammonium acetate (pH 5.6) (1 : l, v/v). No guard column is attached. 20-OH-LTB4 (retention time -- 19.1 -+ 1.6 min; mean -+ SE, n -- 5), 20-CHO-LTB4 (retention time = 17.6 -+ 1.3 rain; mean -+ SE, n = 5), and 20-COOH-LTB4 (retention time = 9.3 -+ 0.5 rain; mean -+ SE, n = 5 (Ref. 10) are eluted in this solvent at a flow rate of 1 ml/min. The 20-CHO-LTB4 peak is collected by hand. Reinjection of a sample of this fraction in the same solvent system demonstrates a single peak. The yield of pure 20CHO-LTB4 is 22.9 -+ 8.3% (mean -+ SD, n = 5). 1° Assay. PMN microsomes (generally 30-75/zl comprising 12.5-75/zg) are incubated in either a plastic Eppendorf or a borosilicate centrifuge tube, with 25 pmol N A D P H and 750 pmol (5/xl of substrate stock) substrate reaction buffer in a final volume of 500/zl. The reaction mixture is then incubated at 37 ° for 20 rain and the reaction terminated by the addition of 500/zl of stop solution. The reaction is then terminated by centrifugation at maximal speed for 5 min in an Eppendorf centrifuge at maximal speed. The supernatant is removed for direct analysis by reversed-phase HPLC. Reversed-Phase HPLC Analysis. Reversed-phase HPLC analysis is performed on a 0.46 × 25-cm, 5/xm analytical column coupled to a 10/xm reversed-phase guard column. The initial solvent is 53% methanol, 47% 50 mM ammonium acetate, pH 5.6 (v/v). The column is equilibrated at a flow rate of 1.0 ml/ml in this buffer. 20-COOH-LTB4 elutes first at 8.0 min, followed by 20-CHO-LTB4 at 16.5 rain and 20-OH-LTB4 at 18 rain (Fig. 1). After 20 min, the solvent composition is altered to 100% methanol. LTB4 is then eluted at 25 rain in a sharp peak. On-line monitoring at 270 nm is performed. The relative areas of the peaks are used to calculate the percentage conversion of substrate to product. When multiplied by the amount of substrate added (750 pmol) the amount of product formed is calculated.
Comments This system allows the measurement of LTB4 and the products of all three co-oxidations catalyzed by cytochrome P-450LTB. The Km for the conversion of LTB4 to 20-OH-LTB4 and 20-OH-LTB4 to 20-CHO-LTB4 have all been reported to be in the range of 0.2-2.0/~m, 7-1° indicating that these reactions are equally efficient. The Vmaxfor both these reactions are reported to be 2400 pmol/min/mg. When 20-CHO is used as a substrate
LTB,
0.05!
A
A2~9 00251
i
, ]
oo 005
i
20 OHLTB ]I t
*
B
i
A~9 0.02~
!
0,0 0,05
~9
0,025
0.0 0.05
Azs9
0,02~
O0
A 0
~0
20
30
RETENTION TIME (m/hutes)
FIG. 1. Metabolism of LTB4 and 20-OH-LTB4 by PMN microsomes. LTB4 (0.6/zM) is incubated alone (A) or with 67.2 (B) or 134.4 (C)/xg o f P M N microsomes together with 100 /xM NADPH for 40 min at 37° in 50 mM Tris-HCl buffer (pH 8.0), and the reaction terminated and analyzed by reversed-phase HPLC in solvent A. 20-OH-LTB4 (1.0/zM) is incubated with 180.0/zg from a second preparation of microsomes (D) for 40 min at 37° in the same buffer, and the reaction terminated and analyzed as described above. Arrows indicate the retention times of chemically synthesized standards and compounds I and II. (Reprinted by permission of the Journal o f Biological Chemistry.)
324
B I O S Y N T H E S I S , E N Z Y M O L O G Y , A N D C H E M I C A L SYNTHESIS
[36]
the Km is lower (0.08/~M) but the Vm~xis markedly less (5 pmol x min -~ x mg-1). Acknowledgment This work was supported by Grants AI-22563, AR-38633 from the National Institutes of Health and in part by a grant-in-aid from the American Heart Association, Massachusetts Affiliate, Inc.
[36] Cytosolic L i v e r E n z y m e s C a t a l y z i n g H y d r o l y s i s of L e u k o t r i e n e A4 to L e u k o t r i e n e B4 and 5 , 6 - D i h y d r o x y e i c o s a t e t r a e n o i c Acid B y JESPER Z. HAEGGSTROM
The unstable allylic epoxide leukotriene (LT) A4 [5(S)-trans-5,6-oxido7,9-trans-1 I, 14-cis-eicosatetraenoic acid] is a key intermediate in the biosynthesis of the biologically active leukotrienes. Enzymatic hydrolysis of LTA4 may be catalyzed by two different enzymes with several functional and structural differences) Thus, LTA4 hydrolase (EC 3.3.2.6) converts the epoxide into LTB4, whereas cytosolic epoxide hydrolase (EC 3.3.2.3) generates 5(S),6(R)-dihydroxy-7,9-trans11,14-cis-eicosate-traenoic acid (5,6-DiHETE).2 Epoxide hydrolases, microsomal or cytosolic, are believed to be involved in detoxification of various harmful xenobiotic epoxides. Although this chapter is primarily concerned with LTA4 hydrolase and cytosolic epoxide hydrolase from guinea pig and mouse liver, respectively, the methods have a broader application since extended studies with synthetic LTA4 and xenobiotic epoxides have shown that both enzymatic activities have a widespread occurrence in mammalian tissues. 3-5 J. Haeggstr6m, J. Meijer, and O. RAdmark, J. Biol. Chem. 261, 6332 (1986). 2 j. Haeggstr6m, A. Wetterholm, M. Hamberg, J. Meijer, R. Zipkin, and O. R~tdmark, Biochim. Biophys. Acta 958, 469 (1988). 3 T. Izumi, T. Shimizu, Y. Seyama, N. Ohishi, and F. Takaku, Biochem. Biophys. Res. Commun. 135, 139 (1986). 4 j. F. Medina, J. Haeggstr6m, M. Kumlin, and O. R~tdmark, Biochim. Biophys. Acta 961, 203 (1988). 5 S. S. Gill and B. D. Hammock, Biochem. Pharmacol. 29, 389 (1980).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All fights of reproduction in any form reserved.
[35]
LTB4
319
5-1ipoxygenase may undergo a product-mediated oxidative activation step; however, the chemical mechanism of such a step remains to be elucidated. As previously mentioned, 5-1ipoxygenase undergoes firstorder irreversible inactivation during the course of its reaction. 4'5 Interestingly, a similar enzyme inactivation occurs upon the exposure of the enzyme to fatty acid hydroperoxide in the absence of substrate turnover. It is possible, therefore, that the product-activated form of the enzyme is unstable. Studies have been performed on the effects of various stimulatory factors on human 5-1ipoxygenase kinetics. 5 These have shown that ATP, the 100,000 g pellet, and the 60-90% precipitate all stimulate the initial velocity of the reaction, and that the increased total product synthesis induced by none of these factors is due to a decreased rate of enzyme inactivation. Interestingly, the 60-90% precipitate actually increases the enzyme inactivation rate as well as the initial velocity. The effects of ATP are not influenced by temperature, but those of both the 100,000 g pellet and the 60-90% precipitate are augmented with increasing temperature. Much remains to be determined concerning the regulation and activation of 5-1ipoxygenase in the intact leukocyte. However, recent evidence indicating that a Ca2÷-dependent association of the enzyme with membrane occurs in ionophore-challenged leukocytes suggests possible roles for both Ca 2+ and membrane in the regulation of 5-1ipoxygenase activity. ~0,~8 Acknowledgments The authors acknowledge the contributions of many friends and colleagues at the Karolinska Institute and the Merck Frosst Centre for Therapeutic Research. We also gratefully acknowledge the support of the Swedish Medical Research Council (Grant # 03X-217), and we thank Ms. Barbara Pearce for excellent secretarial services. t8 C. A. Rouzer and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 84, 7933 (1987).
[35] C y t o c h r o m e P-450LTI3 a n d I n a c t i v a t i o n o f Leukotriene B 4
By RoY J. SOBERMAN Introduction
5(S),12(R)-Dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid [leukotriene B4 (LTB4)] is generally considered the most potent chemotactic compound yet described for human polymorphonuclear leukoMETHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990 by Academic Press, Inc. All fightsof reproductionin any formreserved.
320
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[35]
cytes (PMN). The biological functions of chemotaxis and receptor binding of LTB4 are inactivated by progressive oxidation of C:20.1-4 The o~-oxidation of this pathway is initiated by the enzymatic action of P'450LTB, a cytochrome P-450 mixed-function oxidase located, apparently exclusively, in human PMN. 5-1° Cytochrome P'450LTB is distinct from the oJ-hydroxylases which o~-oxidize prostaglandins and lauric acid by both its substrate specificity9 and lack of immunological crossreactivity with polyclonal antibody to both these enzymes. 9 Cytochrome P-450LTB shows a highly restricted substrate specificity, requiring the "correct" chirality of both the 5- and 12-hydroxyl group configuration and the cis and trans configuration of the double bonds and hydroxyl groups for maximal substrate activity. Cytochrome P-450LTB catalyzes the following reactions: LTB4 + Oz + NADPH + H + ~ 20-OH-LTB4 + NADP ÷ + HzO H 20-OH-LTB4 - (R-C-OH) + NADPH + 02
I
n
(1)
~ 20-CHO (20-aldehyde) - LTB4
o
II (R-C-H) + NADP ÷ + H20
(2)
The overall reaction catalyzed by this enzyme is LTB4 + 2NADPH + 202 ~ 20-CHO-LTB4 + 2NAD + + 2H20
The second reaction, the apparent direct enzymatic oxidation of 20OH-LTB4 by a cytochrome P-450, actually results from the enzymatic addition of a second hydroxyl group followed by the spontaneous dehydration of a dihydroxylated intermediate to an aldehyde. 20-CHO-LTB4 can also be hydroxylated to 20-COOH-LTB4 by cytochrome P'450LTB, but this reaction is relatively slow. l G. Hansson, J. ~. Lindgren, S.-E. Dahl6n, P. Hedqvist, and B. Samuelsson, FEBS Lett. 130, 107 (1981). 2 W. Jubiz, O. Rhdmark, C. Malmsten, G. Hansson, J./~. Lindgren, J. P~lmblad, A.-M. Ud~n, and B. Samuelsson, J. Biol. Chem. 257, 6106 (1982). 3 S. Shak and I. M. Goldstein, J. Biol. Chem. 259, 10181 (1984). 4 W. S. Powell, J. Biol. Chem. 259, 3082 (1984). H. Sumimoto, K. Takeshige, H. Sakai, and S. Minakami, Biochem. Biophys. Res. Commun. 125, 615 (1984). 6 S. Shak and I. M. Goldstein, Biochem. Biophys. Res. Commun. 123, 475 (1984). 7 S. Shak and I. M. Goldstein, J. Clin. Invest. 76, 1218 (1985). 8 R. J. Soberman, T. W. Harper, R. C. Murphy, and K. F. Austen, Proc. Natl. Acad. Sci. U.S.A. 82, 2292 (1985). 9 R. J. Soberman, R. T. Okita, B. Fitzsimmons, J. Rokach, B. Spur, and K. F. Austen, J. Biol. Chem. 262, 12421 (1987). lo R. J. Soberman, J. P. Sutyak, R. T. Okita, D. F. Wendelborn, L. J. Roberts II, and K. F. Austen, J. Biol. Chem. 2.63, 7996 (1988).
[35]
C Y T O C H R O MP-450LTB E AND INACTIVATION OF
LTB4
321
Principle. The conversion of LTB4 to 20-OH-LTB4 and the conversion of 20-OH-LTB4 to 20-CHO-LTB4 and 20-COOH-LTB4 is measured by reversed-phase high-performance liquid chromatography (RP-HPLC) at 280 nm and quantitated by integrated absorbance. Procedures
Reagents. LTB4 and 20-OH LTB4 can be purchased from either Cayman Chemical Company Corporation (Ann Arbor, MI) or Biomol Corporation (Philadelphia, PA). NADP and NADPH are available from Sigma (St. Louis, MO). Methanol HPLC (grade) is obtained from Burdick and Jackson. Acetic acid (reagent grade) and ammonium acetate (HPLC grade) are purchased from Fischer Chemical Corporation. PMN and PMN microsomes are prepared exactly as described by Soberman et al. 9'1° and in this series. ~ Stock Solutions Reaction buffer, 50 mM Tris-HC1, pH 8.0 NADPH, 1.0 mM in Buffer A Substrate, 150/~M LTB4 or 20-OH-LTB4 in ethanol PMN microsomes, generally 0.5-1.0 mg/ml
Preparation of 20-CHO LTB4 Approximately 500 units of equine alcohol dehydrogenase are diluted to 200 ml with 50 mM Tris-HCl buffer (pH 7.0) and dialyzed against 6 liters of this buffer for 48 h to remove the ethanol present in the commercial enzyme preparation. 20-OH-LTB4 (100 lzg) in methanol is then evaporated to dryness under nitrogen in a 250-ml beaker and then resuspended in 80 ml of 0.2 M glycine buffer (pH 10.0; the optimal buffer for alcohol dehydrogenase). NAD + is then added to a concentration of 500/zM, and 54 units of the dialyzed alcohol dehydrogenase added to initiate the reaction. The reaction is allowed to proceed for 90 min at room temperature and then terminated by the addition of 100 ml of stop solution. Twenty 10-ml aliquots are removed, diluted to a volume of 50 ml with cold (4°) distilled water (pH 5.6), and separately concentrated by application onto a single reversed-phase Sep-Pak column (Waters Associates, Milford, MA) previously prepared by washing with 20 ml of methanol, followed by 20 ml of distilled water. The Sep-Pak column is then washed with 20 ml of water, adjusted to pH 5.6 with 0.6 N NaOH, and substrate and products eluted with 2 ml of methanol. The methanol eluate fraction is evaporated to a H R. J. Soberman and R. T. Okita, this series, Vol. 163, p. 349.
322
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[35]
volume of 0.75 ml under nitrogen, mixed with an equal volume of 50 mM ammonium acetate (pH 5.6), and injected onto a 5-/~m reversed-phase HPLC column (0.46 x 25 cm) equilibrated in a solvent of methanol/50 mM ammonium acetate (pH 5.6) (1 : l, v/v). No guard column is attached. 20-OH-LTB4 (retention time -- 19.1 -+ 1.6 min; mean -+ SE, n -- 5), 20-CHO-LTB4 (retention time = 17.6 -+ 1.3 rain; mean -+ SE, n = 5), and 20-COOH-LTB4 (retention time = 9.3 -+ 0.5 rain; mean -+ SE, n = 5 (Ref. 10) are eluted in this solvent at a flow rate of 1 ml/min. The 20-CHO-LTB4 peak is collected by hand. Reinjection of a sample of this fraction in the same solvent system demonstrates a single peak. The yield of pure 20CHO-LTB4 is 22.9 -+ 8.3% (mean -+ SD, n = 5). 1° Assay. PMN microsomes (generally 30-75/zl comprising 12.5-75/zg) are incubated in either a plastic Eppendorf or a borosilicate centrifuge tube, with 25 pmol N A D P H and 750 pmol (5/xl of substrate stock) substrate reaction buffer in a final volume of 500/zl. The reaction mixture is then incubated at 37 ° for 20 rain and the reaction terminated by the addition of 500/zl of stop solution. The reaction is then terminated by centrifugation at maximal speed for 5 min in an Eppendorf centrifuge at maximal speed. The supernatant is removed for direct analysis by reversed-phase HPLC. Reversed-Phase HPLC Analysis. Reversed-phase HPLC analysis is performed on a 0.46 × 25-cm, 5/xm analytical column coupled to a 10/xm reversed-phase guard column. The initial solvent is 53% methanol, 47% 50 mM ammonium acetate, pH 5.6 (v/v). The column is equilibrated at a flow rate of 1.0 ml/ml in this buffer. 20-COOH-LTB4 elutes first at 8.0 min, followed by 20-CHO-LTB4 at 16.5 rain and 20-OH-LTB4 at 18 rain (Fig. 1). After 20 min, the solvent composition is altered to 100% methanol. LTB4 is then eluted at 25 rain in a sharp peak. On-line monitoring at 270 nm is performed. The relative areas of the peaks are used to calculate the percentage conversion of substrate to product. When multiplied by the amount of substrate added (750 pmol) the amount of product formed is calculated.
Comments This system allows the measurement of LTB4 and the products of all three co-oxidations catalyzed by cytochrome P-450LTB. The Km for the conversion of LTB4 to 20-OH-LTB4 and 20-OH-LTB4 to 20-CHO-LTB4 have all been reported to be in the range of 0.2-2.0/~m, 7-1° indicating that these reactions are equally efficient. The Vmaxfor both these reactions are reported to be 2400 pmol/min/mg. When 20-CHO is used as a substrate
LTB,
0.05!
A
A2~9 00251
i
, ]
oo 005
i
20 OHLTB ]I t
*
B
i
A~9 0.02~
!
0,0 0,05
~9
0,025
0.0 0.05
Azs9
0,02~
O0
A 0
~0
20
30
RETENTION TIME (m/hutes)
FIG. 1. Metabolism of LTB4 and 20-OH-LTB4 by PMN microsomes. LTB4 (0.6/zM) is incubated alone (A) or with 67.2 (B) or 134.4 (C)/xg o f P M N microsomes together with 100 /xM NADPH for 40 min at 37° in 50 mM Tris-HCl buffer (pH 8.0), and the reaction terminated and analyzed by reversed-phase HPLC in solvent A. 20-OH-LTB4 (1.0/zM) is incubated with 180.0/zg from a second preparation of microsomes (D) for 40 min at 37° in the same buffer, and the reaction terminated and analyzed as described above. Arrows indicate the retention times of chemically synthesized standards and compounds I and II. (Reprinted by permission of the Journal o f Biological Chemistry.)
324
B I O S Y N T H E S I S , E N Z Y M O L O G Y , A N D C H E M I C A L SYNTHESIS
[36]
the Km is lower (0.08/~M) but the Vm~xis markedly less (5 pmol x min -~ x mg-1). Acknowledgment This work was supported by Grants AI-22563, AR-38633 from the National Institutes of Health and in part by a grant-in-aid from the American Heart Association, Massachusetts Affiliate, Inc.
[36] Cytosolic L i v e r E n z y m e s C a t a l y z i n g H y d r o l y s i s of L e u k o t r i e n e A4 to L e u k o t r i e n e B4 and 5 , 6 - D i h y d r o x y e i c o s a t e t r a e n o i c Acid B y JESPER Z. HAEGGSTROM
The unstable allylic epoxide leukotriene (LT) A4 [5(S)-trans-5,6-oxido7,9-trans-1 I, 14-cis-eicosatetraenoic acid] is a key intermediate in the biosynthesis of the biologically active leukotrienes. Enzymatic hydrolysis of LTA4 may be catalyzed by two different enzymes with several functional and structural differences) Thus, LTA4 hydrolase (EC 3.3.2.6) converts the epoxide into LTB4, whereas cytosolic epoxide hydrolase (EC 3.3.2.3) generates 5(S),6(R)-dihydroxy-7,9-trans11,14-cis-eicosate-traenoic acid (5,6-DiHETE).2 Epoxide hydrolases, microsomal or cytosolic, are believed to be involved in detoxification of various harmful xenobiotic epoxides. Although this chapter is primarily concerned with LTA4 hydrolase and cytosolic epoxide hydrolase from guinea pig and mouse liver, respectively, the methods have a broader application since extended studies with synthetic LTA4 and xenobiotic epoxides have shown that both enzymatic activities have a widespread occurrence in mammalian tissues. 3-5 J. Haeggstr6m, J. Meijer, and O. RAdmark, J. Biol. Chem. 261, 6332 (1986). 2 j. Haeggstr6m, A. Wetterholm, M. Hamberg, J. Meijer, R. Zipkin, and O. R~tdmark, Biochim. Biophys. Acta 958, 469 (1988). 3 T. Izumi, T. Shimizu, Y. Seyama, N. Ohishi, and F. Takaku, Biochem. Biophys. Res. Commun. 135, 139 (1986). 4 j. F. Medina, J. Haeggstr6m, M. Kumlin, and O. R~tdmark, Biochim. Biophys. Acta 961, 203 (1988). 5 S. S. Gill and B. D. Hammock, Biochem. Pharmacol. 29, 389 (1980).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All fights of reproduction in any form reserved.
