Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11603-11606, December 1992 Biochemistry
Purification of human leukotriene C4 synthase (cysteinyl leukotrienes/myeloid cells/glutathione S-transferases)
JOHN F. PENROSE*t, LYNE GAGNON*t, MARGARET GOPPELT-STRUEBE*t, PAUL MYERS*, BING K. LAM*t, RICHARD M. JACK*t, K. FRANK AUSTEN*t, AND RoY J. SOBERMAN*t* *Department of Rheumatology and Immunology, Brigham and Women's Hospital, and the MA 02115
tDepartment of Medicine, Harvard Medical School, Boston,
Contributed by K. Frank Austen, September 4, 1992
ABSTRACT Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexylglutathione-agarose column that was eluted by the use of7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from -75 mg of S-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder of the eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only -1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.
The formation of leukotriene (LT) C4 from membranederived arachidonic acid is catalyzed by three successive enzymatic steps after transmembrane activation of eosinophils, basophils, mast cells, and monocyte/macrophages (1). Arachidonic acid is released from cell membranes by the action of phospholipase A2 (2-5). Then, 5-lipoxygenase is activated independently via a 5-lipoxygenase-associated protein and Ca2+ and catalyzes two sequential enzymatic reactions to form LTA4 (6-10). LTA4 can be converted to LTB4 by the enzyme LTA4 hydrolase (11, 12) or by the enzyme LTC4 synthase to form LTC4 (13-15). Whereas high molecular weight phospholipase A2, 5-lipoxygenase, and LTA4 hydrolase have each been purified and their respective cDNAs cloned (4, 5, 12, 16), LTC4 synthase neither has been purified to homogeneity nor has its cDNA been cloned. The conjugation of reduced glutathione (GSH) with different substrates is catalyzed by the family of enzymes known as glutathione S-transferases (17). These enzymes exist as cytosolic (17) and microsomal forms (18-21). Cytosolic glutathione S-transferases are either homo- or heterodimers of subunits composed of -25 kDa and are traditionally distinguished by their ability to conjugate GSH with various substrates, including 1-chloro-2,4-dinitrobenzene (CDNB) (17). Human and rat liver microsomes (18-21) as well as rat basophil leukemia cell line (RBL-1) cells and guinea pig lung The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11603
(13, 14) have been shown to contain a microsomal glutathione S-transferase that catalyzes the conjugation of CDNB with GSH. This enzyme has been purified from human and rat liver, has been shown to have apparent molecular masses of -17.3 kDa and 14 kDa, respectively, and is encoded by cDNAs of 909 and 883 base pairs, respectively. The capacity to catalyze the conjugation of LTA4 with GSH to form LTC4 has been localized to microsomes of RBL-1 cells (13) and guinea pig lung (14). When RBL-1 microsomes or guinea pig lung microsomes were solubilized utilizing either Triton X-100 or a combination of Triton X-102 and sodium deoxycholate (DOC), the xenobiotic conjugating microsomal glutathione S-transferase was separated chromatographically from LTC4 synthase by either anionexchange chromatography or gel filtration chromatography (13, 14). By these criteria, LTC4 synthase was established as a microsomal enzyme that selectively catalyzes the conjugation of GSH with LTC4. We now report the purification to homogeneity of human LTC4 synthase and its identification as an 18-kDa protein, suggesting that it is a member of the family of microsomal glutathione S-transferases.
MATERIALS AND METHODS Reagents. The following were obtained from suppliers as noted: 2-mercaptoethanol (2-ME), Triton X-102, DOC, Hepes, 3-[cyclohexamino]-1-propanesulfonic acid, glutathione, probenecid, S-hexylglutathione, S-hexylglutathioneagarose, silver nitrate, thioglycolic acid, silver stain kit, and Tris (Sigma); glycerol (EM Science, Gibbstown, NJ); acetic acid, ammonium hydroxide, ammonium acetate (HPLC grade), and disodium EDTA (Fisher); methanol and acetonitrile (Burdick and Jackson); protein determination dye concentrate, acrylamide and bisacrylamide, SDS, and glycine (Bio-Rad); precast Tris-glycine gels (Schleicher & Schuell); and microconcentrators (Amicon and Filtron). LTA4-methyl ester (me) was purchased from Biomol (Plymouth Meeting, PA) and also obtained as a gift from Merck Frosst Labs (Pointe Claire, PQ, Canada). Determination of Enzyme Activities. LTC4 synthase activity was assayed by the conversion of LTA4-me to LTC4-me. LTA4-me was selected as the substrate, rather than LTA4, because of its higher Vma, for conversion to LTC4-me (14). Ten microliters of 2 mM LTA4-me (40 ,uM final concentration) was incubated for 10 min at room temperature in 50 mM Hepes buffer (pH 7.6) with 10 Al of 0.5 M GSH (10 mM final concentration), 10 A.l of 2.5% Triton X-102, and 20 A.l of 0.5 M MgCl2 (20 mM final concentration) and enzyme source to bring the final volume to 500 Al. The MgCl2 was omitted from Abbreviations: 2-ME, 2-mercaptoethanol; CDNB, 1-chloro-2,4dinitrobenzene; DOC, deoxycholate; GS-DNP, S-dinitrophenyl glutathione; GSH, reduced glutathione; LT, leukotriene; me, methyl ester; PG, prostaglandin; RBL-1, rat basophil leukemia cell line. tTo whom reprint requests should be addressed at: Seeley G. Mudd Building, Room 610, 250 Longwood Avenue, Boston, MA 02115.
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the assays conducted in the preliminary purification. Reactions were terminated by the addition of 900 Al of stop solution [methanol/50 mM Hepes, 65:35 (vol/vol)] and 100 1.l of a stock solution (5 Ag/ml) of prostaglandin (PG) B2 as an internal standard. The samples were injected onto a C18 RP-HPLC (0.46 x 25 cm) 5-gm Beckman-Altex ODS column with a 5-,um C18 guard. The column had been preequilibrated in a solvent consisting of methanol/acetonitrile/50 mM ammonium acetate, pH 5.6 (60:16:24, vol/vol). LTs were eluted isocratically at 1.5 ml/min using Beckman model 114 pumps. The formation of LTC4-me was detected by on-line monitoring at 280 nm with a Beckman model 167 scanning UV detector. LTC4-me was quantitated by integrated optical density by comparing the peak areas of LTC4-me and PGB2 and correcting for the molar extinction coefficients of LTC4-me and PGB2. The retention times were as follows: LTC4-me, 5.7 min (range, 5.7-5.9), and PGB2, 3.4 min (range,
3.3-3.6). Glutathione S-transferase activity was assayed by the conversion of CDNB to S-dinitrophenyl glutathione (GSDNP). Ten microliters of 100 mM CDNB (2 mM final concentration) was incubated for 15 min at 22°C in 50 mM K3PO4 buffer (pH 6.5) with 10 mM GSH and enzyme source to bring the final volume to 500 ,l. Reactions were terminated by the addition of 900 Al of stop solution consisting of methanol/acetic acid (50:1, vol/vol) and 100 ,.l of PGB2 (5 ,g/ml) as an internal standard. Samples were routinely centrifuged at 10,000 x g to remove precipitated proteins. The samples were analyzed by RP-HPLC with on-line monitoring at 340 nm and 280 nm simultaneously for GS-DNP and PGB2 as described (22). PGB2 and GS-DNP were quantitated using integrated optical densities, comparing the peak area of GS-DNP and PGB2 and correcting for their respective molar extinction coefficients. Retention times were 7.1 min and 20.1 min for GS-DNP and PGB2, respectively. Protein Determination and Electrophoresis. Protein was measured using a modification of the Bradford technique with reagents from Bio-Rad (23). SDS/PAGE with 14% Trisglycine gels was performed according to the method of Laemmli (24). Proteins within gels were detected using silver stains.
Preparation of KG-1 Microsomes. The myelomonocytic leukemia cell line KG-1 was obtained from the American Type Culture Collection. Cells were grown by the National Cell Culture Center (Minneapolis), in RPMI medium supplemented with 6% calf serum and 4% fetal bovine serum, 10 mM Hepes buffer (pH not adjusted), and 1% penicillin/ streptomycin solutions. The cells were seeded at a density of 1 x 104 per ml in 8-liter spinner flasks and grown to a maximum density of 1-1.5 x 106 per ml. From pooled 8-liter spinner flasks, a total of 30 liters of cells was harvested by centrifugation at 1000 x g for 10 min at 4°C, washed in a small amount of 50 mM Hepes/5 mM 2-ME/1 mM EDTA, pH 7.6, and shipped on dry ice. Microsomes were prepared by resuspending the cells in 400 ml of buffer containing 50 mM Hepes, 0.25 M sucrose, 5 mM 2-ME, 1 mM EDTA, and 1o glycerol (pH 7.6) and then sonicating the cells on ice for five 3-min pulses (60%o work cycle, setting 6) with a Branson sonifier. The sonicate was centrifuged in 250-ml conical tubes at 1000 x g for 10 min at 4°C to sediment cell debris and unbroken cells. The supernatants containing the microsomes were cleared by centrifugation at 10,000 x g at 4°C for 15 min in 50-ml Sorvall centrifuge tubes and were decanted into 30-ml ultracentrifuge tubes. After centrifugation at 100,000 x g at 4°C for 1 hr, the supernatants were removed and the pellets containing microsomes were washed in the same buffer and centrifuged again at 100,000 x g at 40C for 1 hr. Solubilization was performed by pooling all pellets and gently stirring the microsomes into a buffer containing 0.4% DOC, 0.4% Triton X-102, and 10% glycerol for 1 hr at 40C. The
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Proc. Natl. Acad. Sci. USA 89 (1992)
solubilized microsomes were centrifuged at 100,000 x g for 1 hr at 40C, and the supernatants (solubilized microsomes) were frozen at -70'C.
RESULTS S-Hexylglutathione-Agarose Chromatography of LTC4 Synthase in Solubilized Microsomes. Two 30-liter batches of KG-1 cells were utilized to prepare and solubilize 1265 mg of microsomes as described. A 5.0 x 2.5 cm column of S-hexylglutathione-agarose was equilibrated at 40C in a buffer containing 50 mM Hepes, 1 mM EDTA, 5 mM 2-ME, 0.1% Triton X-102, and 1o glycerol (pH 7.6) (buffer A). One hundred fifteen milliliters of the solubilized microsomes was applied at a flow rate of 45 ml/hr, the flow-through was collected in a single batch and then reapplied at 45 ml/hr, and the flow-through was again collected. The column was washed with 145 ml of buffer A, containing 0.3 M NaCI, 0.1% DOC, and 20 mM GSH, and then washed with 100 ml of buffer A with 0.1% DOC. The column was batch eluted with 105 ml of buffer A with 7.5 mM probenecid and 0.1% DOC, followed by additional elution with buffer A containing 30 mM probenecid and 0.1% DOC in 8-ml fractions. As shown in Fig. 1, all of the LTC4 synthase activity bound to the column; none was found in the fall-through or the washes. Forty-nine percent of the activity applied to the column was eluted by the addition of 7.5 mM probenecid and 8% in the subsequent fractions eluted with 30 mM probenecid. No glutathione S-transferase activity was detected either in the solubilized microsomes or in any column fractions. The enzyme activity eluted by the addition of 7.5 mM probenecid was concentrated in an Amicon concentrator at 4°C, from a volume of 105 ml to 50 ml. The concentrate was then rediluted to a volume of 75 ml with detergent-free buffer A, followed by four sequential 2-fold concentrations using the Amicon concentrator and redilution to one-half the preceding volume. Removal of probenecid and concentration of volume were necessary for further chromatographic steps. The overall recovery of LTC4 synthase during concentration and .f...e.B.
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FIG. 1. S-Hexylglutathione-agarose column chromatography. One hundred fifteen milliliters of solubilized microsomes was applied to the column and successively washed with buffer A containing 0.3 M NaCl, 0.1% DOC, and 20 mM GSH (wash 1) and buffer A containing 0.1% DOC (wash 2); elution was then carried out with buffer A containing 7.5 mM probenecid and 0.1% DOC (elution 1) and buffer A containing 30 mM probenecid and 0.1% DOC (elution 2). Only the initial fraction of six fractions of elution 2 is depicted.
dilution was 118%6. The final 11.5-ml concentrate was frozen at -700C for 12 hr with no loss of enzyme activity. S alexy~hdaddone-Aprese Cb I Ih- of PartaLy Puried LTC4 Synthme. The concentrated enzyme was quickly thawed at 370C and mixed in a 15-ml polypropylene tube with 75 mg of S-hexylglutathione-agarose preequilibrated in buffer A and placed on a rocking shaker for 90 min at 40C. The mixture was centrifuged at 500 x g for S nmn, and the resin was washed two times at 5-min intervals with a total of 8 ml of buffer A with 0.3 M NaCl, 0.1% DOC, and 20 mM GSH and 4.5 ml of buffer A containing 10 mM S-hexylglutathione with 10 mM GSH. The resin was then batch eluted by the addition of 3.5 ml of buffer A with 7.5 mM probenecid and 0.1% DOC and transferred to a spin column for additional elutions. The latter were performed by adding 200 1lI of buffer A with 30 mM probenecid and 0.1% DOC to the resin, centrifuging for 10 sec in an Eppendorf centrifuge, and collecting the wash in the lower tube. This procedure was repeated six times. Aliquots were assayed and the remainders of the washes and eluates were frozen at -700C. Eighty-three percent of the enzyme activity bound to the resin, and none was eluted in either of the first two washes. Of the added enzyme, 14% was batch eluted in the 7.5 mM probenecid fraction and an additional 9% was eluted in the first three 30 mM probenecid fractions. Native Gel Electrophureuis. The 7.5 mM probenecid eluate from the small-batch S-hexyl agarose step was concentrated to 110 Al& using a Centricon spin concentrator with 10-kDa cutoff size by centrifugation at 5000 x g for 45 min at 40C. Twenty-five microliters of non-SDS-containing gel loading buffer with 2 mM thioglycolic acid was added to the concentrate. A non-SDS 4-12% Tris-glycine gel was prerun in a non-SDS buffer of 100 mM Tris, 1 mM EDTA, 2 mM thioglycolic acid, 0.1% Triton, and 0.1% DOC at pH 9.2 at 25 mA with unlimited voltage for 30 min at 40C, and 80 ,l of the concentrate containing partially purified LTC4 synthase was then loaded into the two center lanes of this prerun gel. The gel was electrophoresed in the same buffer for 6.5 hr at 20 mA with unlimited voltage at 4C. The gel was removed and sliced into 0.5-cm fractions, which were individually eluted by vigorous shaking in 500 ;l of 50 mM Hepes buffer with 1 mM EDTA, 5 mM 2-ME, 0.1% Triton X-102, 10%o glycerol, 0.7 M NaCl, and 20 mM GSH at pH 7.6 for 15 hr at 40C. Twenty-five microliters of eluate was assayed for LTC4 synthase activity, and the remaining =375 ;LI recovered was precipitated in 1.8-ml prechilled acetone for 1 hr at -200C and centrifuged at 10,000 x g for 10 min. The pellets were dried, resuspended in 35 ul of SDS gel loading buffer, heated to 1000C for 5 min, and analyzed by SDS/PAGE in a 14% precast Tris-glycine gel followed by silver staining. As shown in Fig. 2, enzyme activity eluted in fractions 3 and 4. Thirty-eight percent of the enzyme activity loaded into the gel was recovered in lane 3, and 84% was recovered in lane 4, indicating no loss at this step, with an overall recovery of 12.5% for the entire purification scheme. A single silverstained band of 18 kDa was observed in lane 4. Two additional proteins of 27 kDa and 36 kDa were visible in lane 3 but were absent in lane 4, which contained the predominant LTC4 synthase activity. In a preliminary purification, using steps that had been developed with various other chromatographic sequences, microsomes from 150 liters of KG-1 were applied to an open 15 x 2.5 cm S-hexylglutathione-agarose column and washed with the same buffers described above in 300-ml quantities. The LTC4 synthase activity was not eluted with 300 ml of buffer A containing 0.1% DOC and 7.5 mM probenecid but was eluted in the five 15-mi fractions of buffer A with 30 mM probenecid and 0.1% DOC with a recovery of 62%. The active fractions were pooled and concentrated to 20 ml with an Amicon concentrator and frozen as previously described.
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Proc. Natd. Acad. Sci. USA 89 (1992)
Biochemistry: Penrose et A
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FIG. 2. SDS/PAGE identification of LTC4 syntas. Eluates from seven 0.5-cm slices of a nondenaturing acrylamide gel were assayed for LTCE synthase activity, and the remadnder was precipitated and analyzed by a 14% SDS gel with silver staining for proteins. The LTC4 synthase activity in each assay (one-sixteenth of the eluate) is recorded at the top of each corresponding SDS gel lane.
One-half of the sample (10 ml) was then bound to 100 mg of S-hexylglutathione-agarose and the resin was washed as described above and eluted with six sequential additions of buffer A with 30 mM probenecid and 0.1% DOC in the spin column. The activity was recovered in the first three elutions with a yield of 6.7% and was concentrated 3-fold with a Centricon spin concentrator. The concentrated fraction containing partally purified LTC4 synthase was subjected to native gel electrophoresis. One hundred microliters of the recovered eluate was assayed for function and the remainder was subjected to SDS/PAGE as described. Overall, 87% of the applied LTC4 synthase activity was eluted from gel fractions 6 and 7, with 34% (4.2 nmol per total eluate) and 42% (5.2 nmol per total eluate) recoveries, respectively. When analyzed by SDS/PAGE and silver staining, only bands of 18 kDa were observed in lanes corresponding to the gel slice from which activity had been eluted. This band was not present in any other lane. Overall recovery for this purification was 0.8%.
DISCUSSION LTC4 synthase was solubilized from the microsomes of the myelomonocytic KG-1 cell line using 6 x 1010 starting cells (Fig. 1). The initial S-hexylglutathione-agarose binding step achieved a 1700-fold purification of this enzyme. This is attributed to the elution of contaminating proteins in the wash buffer with 20 mM GSH and the retention of LTC4 synthase until elution with probenecid. The sequential concentration and dilution of the eluate were required to remove probenecid from the enzyme and provided an 8.8-fold concentration of preparation. The subsequent S-hexylglutathione binding step gave an additional 7-fold purification and also served to concentrate the enzyme further as was needed for the subsequent resolution by nondenaturing acrylamide gel electrophoresis. The choice of the acrylamide composition and time of electrophoresis were chosen so that the enzyme migrated 35-500% of the distance into the gel, allowing maximal resolution from other proteins (Fig. 2). The identification of LTC4 synthase as a protein of 18 kDa is based upon the finding that eluates of those gel slices with the predominant LTC4 synthase activity demonstrated a single silver-staining band in
SDS/PAGE.
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The overall recovery for the optimized procedure was 12.5%. The specific activity of the enzyme preparation increased -10,000 fold from the solubilized microsomes to the second S-hexylglutathione-agarose binding step with probenecid elution. The inability to measure protein in eluates of the slices from the nondenaturing gel and the detection of a single silver-staining band would indicate that the final purification was >10,000 fold. The utility ofprobenecid for elution ofLTC4 synthase from the S-hexylglutathione-agarose column was suggested by the ability of this molecule to block the carrier-mediated export of newly synthesized intracellular LTC4 from eosinophils (22, 25). This effect may be related to the structure of probenecid, which contains two hydrocarbon chains linked by sulfur and nitrogen atoms to a phenolic ring. In addition, whereas S-hexylglutathione inhibits LTC4 synthase activity at micromolar concentrations (14), probenecid does not interfere with the conjugation reaction at similar concentrations. The most likely reason for the finding that 7.5 mM probenecid, rather than 30 mM (as suggested by the preliminary experiment), is sufficient to elute LTC4 from the S-hexylglutathione resin would be that the optimal concentration is close to 7.5 mM. As slight differences in the detergent concentration carried forward with the buffer might influence the elution, a higher concentration might prove more reliable as a single elution concentration. The identification of LTC4 synthase as an 18-kDa protein suggests strongly that it is a member of the microsomal glutathione S-transferase family with a restricted substrate specificity and limited cellular distribution. This work was supported in part by Grants AI-22531, AI-22563, AI-26292, HL-36110, AI-31599, ES-05859, and RR-05950 from the National Institutes of Health. J.F.P. was a trainee on Grant T32 AR-07530 from the National Institutes of Health. 1. Lewis, R. A., Austen, K. F. & Soberman, R. J. (1990) N. Engl. J. Med. 323, 645-655. 2. Clark, J. D., Milona, N. & Knopf, J. L. (1990) Proc. Nat!. Acad. Sci. USA 87, 7708-7712. 3. Kramer, R. M., Roberts, E. F., Manetta, J. & Putnam, J. E.
(1991) J. Biol. Chem. 266, 5268-5272.
4. Clark, J. D., Lin, L.-L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N. & Knopf, J. L. (1991) Cell 65, 1043-1051.
Proc. Nad. Acad. Sci. USA 89 (1992) 5. Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Gecker, G. W., Kang, L. H., Roberts, E. F. & Kramer, R. M. (1991) J. Biol. Chem. 266,14850-14853. 6. Shimizu, T., Radmark, 0. & Samuelsson, B. (1984) Proc. Natl. Acad. Sci. USA 81, 689-693. 7. Rouzer, C. A. & Samuelsson, B. (1985) Proc. Natl. Acad. Sci. USA 82, 6040-6044. 8. Rouzer, C. A., Matsumoto, T. & Samuelsson, B. (1986) Proc. Nat!. Acad. Sci. USA 83, 857-861. 9. Miller, D. K., Gillard, J. W., Vickers, P. J., Sadowski, S., Leveille, C., Mancini, J. A., Charleson, P., Dixon, R. A. F., Ford-Hutchinson, A. W., Fortin, R., Gauthier, J. Y., Rodkey, J., Rosen, R., Rouzer, C., Sigal, I. S., Strader, C. D. & Evans, J. F. (1990) Nature (London) 343, 278-281. 10. Dixon, R. A. F., Diehl, R. E., Opas, E., Rands, E., Vickers, P. J., Evans, J. F., Gillard, J. W. & Miller, D. K. (1990) Nature (London) 343, 282-284. 11. Radmark, O., Shimizu, T., Jornvall, H. & Samuelsson, B. (1984) J. Biol. Chem. 259, 12339-12345. 12. Minani, M., Ohno, S., Kawasaki, H., Radmark, O., Samuelsson, B., Jornvall, H., Shimizu, T., Seyama, Y. & Suzuki, K. (1987) J. Biol. Chem. 262, 13873-13876. 13. Yoshimoto, T., Soberman, R. J., Lewis, R. A. & Austen, K. F. (1985) Proc. Nat!. Acad. Sci. USA 82, 8399-8403. 14. Yoshimoto, T., Soberman, R. J., Spur, B. & Austen, K. F. (1988) J. Clin. Invest. 81, 866-871. 15. Soderstrom, M., Mannervik, B., Garkov, V. & Hammarstrom, S. (1992) Arch. Biochem. Biophys. 294, 70-74. 16. Dixon, R. A. F., Jones, R. E., Diehl, R. E., Bennett, C. D., Kargman, S. & Rouzer, C. A. (1988) Proc. Nat!. Acad. Sci. USA 85, 416-420. 17. Jacoby, W. B. (1985) Methods Enzymol. 113, 495-510. 18. Morgenstern, R., Guthenberg, C. & Depierre, J. W. (1982) Eur. J. Biochem. 128, 243-248. 19. McLellan, L. I., Wolf, C. R. & Hayes, J. D. (1989) Biochem. J. 258, 87-93. 20. Morgenstern, R., Depierre, J. W. & Jornvall, H. (1985) J. Biol. Chem. 260, 13976-13983. 21. Dejong, J. L., Morgenstern, R., Jornvall, H. J., Depierre, J. W. & Tu, C. P. (1988) J. Biol. Chem. 263, 8430-8436. 22. Lam, B. K., Owen, W. F., Austen, K. F. & Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889. 23. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 24. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 25. Lam, B. K., Xu, K., Atkins, M. B. & Austen, K. F. (1992) Proc. Nat!. Acad. Sci. USA 89, 11598-11602.