[37]
L T C 4 S Y N T H A S E F R O M G U I N E A PIG L U N G M I C R O S O M E S
335
[37] P u r i f i c a t i o n a n d P r o p e r t i e s o f L e u k o t r i e n e C4 S y n t h a s e from Guinea Pig Lung Microsomes B y RoY J. SOBERMAN
Leukotriene C 4 synthase is the enzyme that catalyzes the conjugation of leukotriene (LT) A4 with reduced glutathione (GSH). This enzyme is a novel member of the family of glutathione S-transferases and is distinct from other cytosolic glutathione S-transferases, and from the microsomal glutathione S-transferase that utilizes 1-chloro-2,4-dinitrobenze as a substrate. It has been partially purified and characterized from rat basophilic leukemia (RBL-1) cell microsomes 1'2 and from guinea pig lung microsome. 3,4 Principle. The conjugation of LTA4 and GSH to form LTC4 is monitored by on-line monitoring at 280 nm and quantitated by integrated absorbance.
Reagents
LTA4-methyl ester (me) can be purchased from either Biomol (Philadelphia, PA) or Cayman Chemical Corp. (Ann Arbor, MI). GSH, prostaglandin (PG) B2, Triton X-102, and glycerol are obtained from Sigma (St. Louis, MO). Reaction Buffer: 50 mM HEPES, pH 7.6 Substrate stock solutions: (a) 100 mM GSH; (b) 200 tzM LTA4-me or LTA4 (prepared by hydrolysis of LTA4-me as previously described 2) Stop solution: Methanol : acetonitrile : acetic acid 50 : 50 : 1 (v/v/v) containing 100/zM PGB2/0.5 ml Reversed-phase HPLC Buffer: (A) methanol : acetonitrile : water : acetic acid, pH 5.6 (13:5:8.1:0.9, v/v/v/v) (for LTA4-me); or (B) methanol : acetonitrile : water : acetic acid, pH 5.6 (5 : 5 : 8.1 : 0.9, v/v/v/v) (for LTA4) Reversed-phase HPLC column (C18): 5/zM, 4.1 x 250 mm with a 10-~m guard column 1M. K. Bach,J. R. Brashler,and D. R.Morton, Jr., Arch. Biochern. Biophys. 230,455 (1984). 2 T. Yoshimoto,R. J. Soberman, R. A. Lewis, and K. F. Austen,Proc. Natl. Acad. Sci. U.S.A. 82, 8399 (1986). 3 T. Yoshimoto,R. J. Soberman, B. Spur, and K. F. Austen,J. Clin. Invest. 81, 866 (1988). 4 T. Izumi, Z. Honda, N. Ohishi, S. Kitamura,S. Tsuchida, K. Sato, T. Shimuzu,and Y. Seyama,Biochim. Biophys. Acta 959, 305 (1988). METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
336
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[37]
Assay Method Enzyme, l0 nmol LTA4 or LTA4-methyl ester are incubated for 10 min in reaction buffer with 5/~mol GSH and enzyme in a 0.5 ml volume at 22 °. Stop solution, 0.5 ml, is added to terminate the reaction and the mixture centrifuged in an Eppendorf microfuge tube. For LTA4-me, the supernatant is analyzed in buffer A at a flow rate of 1 ml/min with on-line monitoring at 280 nm. The retention times of PGBz and LTCa-me are 8.2 min and 12.2 rain, respectively. 3 The total amount of LTC4-me formed is quantified by comparing the ratio of LTC4-me to PGB2 with a standard curve. When LTC4 analysis is performed, the ratio of the integrated area of LTC4 (9.1 min) to PGB2 06.4 min) is calculated. Purification
Preparation of Guinea Pig Lung Microsomes. Lungs are collected in ice-cold 20 mM Tris-HCl buffer, pH 8.0, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM 2-mercaptoethanol. They are then cut into small pieces with scissors. The fragments are rinsed once in the same buffer and then resuspended in 5-volume wet weight of buffer and disrupted using a Tekmar Tissuemizer model SDT- 1810. The homogenate is centrifuged at 1000 g for 10 min to remove nondisrupted tissue and the supernatant filtered through cheesecloth. The filtrate is then centrifuged at 10,000 g for I0 min and the resulting supernatant removed and centrifuged at 105,000 g for 60 min. All centrifugations are carried out at 4 ° and homogenization is performed on ice. The supernatant (cytosol) is removed, the microsomes resuspended and washed once in the same buffer, centrifuged at I05,000 g for I hr, and resuspended in an equal volume of the same buffer. Glycerol is added to microsomes to achieve a concentration of 20% (w/v). To solubilize the enzyme, Triton X-102 and deoxycholate are added dropwise to concentrations of 0.4% each and then stirred for 1 hr at 4 °. The mixture is then centrifuged at 105,000g for 1 hr. Approximately 50% of the activity is recovered in the supernatant. Approximately 35 ml of solubilized enzyme, is applied to a Sepharose CL-4B column (4.4 x 50 cm) equilibrated with 20 mM Tris-HCl buffer at pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, and 10% glycerol containing 0.1% Triton X-102 and 0.1% deoxycholic acid. This mixture of detergent is required to prevent the formation of large aggregates. The column is run at a flow rate of ---45 ml/hr. Microsomal LTC4 synthase is eluted at an apparent size of =55,000 distinguishing it from the microsomal glutathione S-transferase which eluted at a molecular weight of ---34,000.
[37]
LTC4 SYNTHASE FROM
GUINEA PIG LUNG MICROSOMES
337
The peak fractions containing LTC4 synthase activity are pooled and applied to a DEAE-Sephacel column (2.5 x 4 cm) equilibrated with the same buffer. The enzymatic activity is recovered in the flow-through. The pH of the column should be kept below 8.0, as significant amounts of activity will adhere at pH values greater than 8.2. LTC4 synthase appears in the effluent. An approximately 30-fold purification is achieved in this step. The DEAE-Sephacel flow-through is then applied to an agarosebutylamine column (2.5 x 10 cm) previously equilibrated with the same buffer. The column is washed with two column volumes of buffer and then eluted with the same buffer; 6-ml fractions are collected. The pooled enzyme preparation is injected onto a DEAE-3SW HPLC column (2.15 x 15 cm) equilibrated with 50 mM potassium phosphate buffer at pH 8.0 containing I mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, and 0.1% Triton X-102. The column is washed at a flow rate of 3 ml/min and then eluted in a linear gradient (120 ml) of 0 to 0.2 M NaCI and 0 to 0.1% deoxycholic acid in the same buffer. LTC4 synthase elutes at a salt concentration of =0.16 M and a deoxycholate concentration of 0.08%. Peak active fractions are pooled, frozen at - 7 0 ° , and used as the source of purified LTC4 synthase. We have previously achieved an overall purification of -91-fold with a 3% yield. 3 Properties The enzyme has somewhat lower Km for LTA4 (3/zM) than for LTA4me (15/zM), although the Vm~x for LTA4 (108/.~mol/3 rain/rag) is significantly lower than for LTA4-me (420/zmol/min/mg) allowing the latter to be used as a substrate for detection during purification. Both substrates complete with each other, indicating that they are substrates for the same enzyme. LTC4 is an excellent inhibitor of LTC4-me synthase activity. With LTA4-me as a substrate, the ICso for LTCso is 2.1 ;zM. The actual subunit size and composition awaits complete purification. Acknowledgment This work was supported by grants AI22563, AR38638and by a grant in aid from the American Heart Association with funds contributed in part by the Massachusetts Chapter.
L T C 4 S Y N T H A S E F R O M G U I N E A PIG L U N G M I C R O S O M E S
335
[37] P u r i f i c a t i o n a n d P r o p e r t i e s o f L e u k o t r i e n e C4 S y n t h a s e from Guinea Pig Lung Microsomes B y RoY J. SOBERMAN
Leukotriene C 4 synthase is the enzyme that catalyzes the conjugation of leukotriene (LT) A4 with reduced glutathione (GSH). This enzyme is a novel member of the family of glutathione S-transferases and is distinct from other cytosolic glutathione S-transferases, and from the microsomal glutathione S-transferase that utilizes 1-chloro-2,4-dinitrobenze as a substrate. It has been partially purified and characterized from rat basophilic leukemia (RBL-1) cell microsomes 1'2 and from guinea pig lung microsome. 3,4 Principle. The conjugation of LTA4 and GSH to form LTC4 is monitored by on-line monitoring at 280 nm and quantitated by integrated absorbance.
Reagents
LTA4-methyl ester (me) can be purchased from either Biomol (Philadelphia, PA) or Cayman Chemical Corp. (Ann Arbor, MI). GSH, prostaglandin (PG) B2, Triton X-102, and glycerol are obtained from Sigma (St. Louis, MO). Reaction Buffer: 50 mM HEPES, pH 7.6 Substrate stock solutions: (a) 100 mM GSH; (b) 200 tzM LTA4-me or LTA4 (prepared by hydrolysis of LTA4-me as previously described 2) Stop solution: Methanol : acetonitrile : acetic acid 50 : 50 : 1 (v/v/v) containing 100/zM PGB2/0.5 ml Reversed-phase HPLC Buffer: (A) methanol : acetonitrile : water : acetic acid, pH 5.6 (13:5:8.1:0.9, v/v/v/v) (for LTA4-me); or (B) methanol : acetonitrile : water : acetic acid, pH 5.6 (5 : 5 : 8.1 : 0.9, v/v/v/v) (for LTA4) Reversed-phase HPLC column (C18): 5/zM, 4.1 x 250 mm with a 10-~m guard column 1M. K. Bach,J. R. Brashler,and D. R.Morton, Jr., Arch. Biochern. Biophys. 230,455 (1984). 2 T. Yoshimoto,R. J. Soberman, R. A. Lewis, and K. F. Austen,Proc. Natl. Acad. Sci. U.S.A. 82, 8399 (1986). 3 T. Yoshimoto,R. J. Soberman, B. Spur, and K. F. Austen,J. Clin. Invest. 81, 866 (1988). 4 T. Izumi, Z. Honda, N. Ohishi, S. Kitamura,S. Tsuchida, K. Sato, T. Shimuzu,and Y. Seyama,Biochim. Biophys. Acta 959, 305 (1988). METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
336
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[37]
Assay Method Enzyme, l0 nmol LTA4 or LTA4-methyl ester are incubated for 10 min in reaction buffer with 5/~mol GSH and enzyme in a 0.5 ml volume at 22 °. Stop solution, 0.5 ml, is added to terminate the reaction and the mixture centrifuged in an Eppendorf microfuge tube. For LTA4-me, the supernatant is analyzed in buffer A at a flow rate of 1 ml/min with on-line monitoring at 280 nm. The retention times of PGBz and LTCa-me are 8.2 min and 12.2 rain, respectively. 3 The total amount of LTC4-me formed is quantified by comparing the ratio of LTC4-me to PGB2 with a standard curve. When LTC4 analysis is performed, the ratio of the integrated area of LTC4 (9.1 min) to PGB2 06.4 min) is calculated. Purification
Preparation of Guinea Pig Lung Microsomes. Lungs are collected in ice-cold 20 mM Tris-HCl buffer, pH 8.0, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM 2-mercaptoethanol. They are then cut into small pieces with scissors. The fragments are rinsed once in the same buffer and then resuspended in 5-volume wet weight of buffer and disrupted using a Tekmar Tissuemizer model SDT- 1810. The homogenate is centrifuged at 1000 g for 10 min to remove nondisrupted tissue and the supernatant filtered through cheesecloth. The filtrate is then centrifuged at 10,000 g for I0 min and the resulting supernatant removed and centrifuged at 105,000 g for 60 min. All centrifugations are carried out at 4 ° and homogenization is performed on ice. The supernatant (cytosol) is removed, the microsomes resuspended and washed once in the same buffer, centrifuged at I05,000 g for I hr, and resuspended in an equal volume of the same buffer. Glycerol is added to microsomes to achieve a concentration of 20% (w/v). To solubilize the enzyme, Triton X-102 and deoxycholate are added dropwise to concentrations of 0.4% each and then stirred for 1 hr at 4 °. The mixture is then centrifuged at 105,000g for 1 hr. Approximately 50% of the activity is recovered in the supernatant. Approximately 35 ml of solubilized enzyme, is applied to a Sepharose CL-4B column (4.4 x 50 cm) equilibrated with 20 mM Tris-HCl buffer at pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, and 10% glycerol containing 0.1% Triton X-102 and 0.1% deoxycholic acid. This mixture of detergent is required to prevent the formation of large aggregates. The column is run at a flow rate of ---45 ml/hr. Microsomal LTC4 synthase is eluted at an apparent size of =55,000 distinguishing it from the microsomal glutathione S-transferase which eluted at a molecular weight of ---34,000.
[37]
LTC4 SYNTHASE FROM
GUINEA PIG LUNG MICROSOMES
337
The peak fractions containing LTC4 synthase activity are pooled and applied to a DEAE-Sephacel column (2.5 x 4 cm) equilibrated with the same buffer. The enzymatic activity is recovered in the flow-through. The pH of the column should be kept below 8.0, as significant amounts of activity will adhere at pH values greater than 8.2. LTC4 synthase appears in the effluent. An approximately 30-fold purification is achieved in this step. The DEAE-Sephacel flow-through is then applied to an agarosebutylamine column (2.5 x 10 cm) previously equilibrated with the same buffer. The column is washed with two column volumes of buffer and then eluted with the same buffer; 6-ml fractions are collected. The pooled enzyme preparation is injected onto a DEAE-3SW HPLC column (2.15 x 15 cm) equilibrated with 50 mM potassium phosphate buffer at pH 8.0 containing I mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, and 0.1% Triton X-102. The column is washed at a flow rate of 3 ml/min and then eluted in a linear gradient (120 ml) of 0 to 0.2 M NaCI and 0 to 0.1% deoxycholic acid in the same buffer. LTC4 synthase elutes at a salt concentration of =0.16 M and a deoxycholate concentration of 0.08%. Peak active fractions are pooled, frozen at - 7 0 ° , and used as the source of purified LTC4 synthase. We have previously achieved an overall purification of -91-fold with a 3% yield. 3 Properties The enzyme has somewhat lower Km for LTA4 (3/zM) than for LTA4me (15/zM), although the Vm~x for LTA4 (108/.~mol/3 rain/rag) is significantly lower than for LTA4-me (420/zmol/min/mg) allowing the latter to be used as a substrate for detection during purification. Both substrates complete with each other, indicating that they are substrates for the same enzyme. LTC4 is an excellent inhibitor of LTC4-me synthase activity. With LTA4-me as a substrate, the ICso for LTCso is 2.1 ;zM. The actual subunit size and composition awaits complete purification. Acknowledgment This work was supported by grants AI22563, AR38638and by a grant in aid from the American Heart Association with funds contributed in part by the Massachusetts Chapter.
