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Enzymes for anandamide biosynthesis and metabolism
Jou|~t of
LIPID MEDIATORS ANIDC|LL SIGNALLING
ELSEVIER
J. Lipid Mediators Cell Signalling 14 (1996) 57 61
Enzymes for anandamide biosynthesis and metabolism Natsuo Ueda a, Yuko KurahashP, Kei Yamamoto ~, Shozo Yamamoto ~'*, Takashi Tokunaga b aDepartment of Biochemistry, Tokushima University, School of Medicine, Tokushima 770, Japan bJapan Tobacco Inc., Central Pharmaceutical Research Institute, Takatsuki, Osaka 569, Japan
Abstract Anandamide is an endogenous ligand for cannabinoid receptors. We tried to isolate and purify 'anandamide amidohydrolase' which hydrolyzes anandamide to arachidonic acid and ethanolamine. The enzyme activity was found in the microsomal fraction of porcine brain homogenate. The enzyme was solubilized in 1% Triton X-100, and partially purified by hydrophobic chromatography to a specific activity of about 0.3 /~mol/min per mg protein (37°C). Apparent K m for anandamide was about 60 /tM. The enzyme reacted also with ethanolamides of linoleic, oleic, and palmitic acids at lower rates. This enzyme preparation also converted arachidonic acid to anandamide in the presence of 250 mM concentration of ethanolamine. Several lines of evidence including experiments using various inhibitors suggested that the anandamide synthase and amidohydrolase activities were derived from a single enzyme protein. Keywords: Anandamide; Cannabinoid; Lipoxygenase
1. Introduction F o r a better u n d e r s t a n d i n g of the physiological f u n c t i o n of a n a n d a m i d e , a n e n d o g e n o u s ligand for c a n n a b i n o i d receptors ( D e v a n e et al., 1992), we m u s t
* Corresponding author. Tel.: + 81 886 337058; fax: + 81 886 336409. 0929-7855/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0929-7855(96)00509-3
58
N. Ueda et al. / J. Lipid Mediators Cell Signalling 14 (1996) 57 61
elucidate how the production and degradation of this new compound are enzymatically controlled in the cells. As shown in Fig. 1, it was suggested that a phospholipase D was activated and released anandamide from N-arachidonylethanolamide phospholipids (Di Marzo et al., 1994; Sato et al., 1994). The bioactive anandamide was then hydrolyzed and inactivated by a hydrolase (Deutsch and Chin, 1993; Desarnaud et al., 1995). In reverse, anandamide was synthesized by the condensation of arachidonic acid with ethanolamine (Devane and Axelrod, 1994; Kruszka and Gross, 1994). However, the enzymes responsible for these interconversions have not been clearly characterized on the enzymological and molecular biological levels. In this paper we report partial purification and characterization of 'anandamide amidohydrolase' of porcine brain.
2. Materials and methods
The microsomal fraction of porcine brain was suspended in 50 m M Tris-HC1 buffer (pH 8) containing 1% Triton X-100, kept for 12 h, and then centrifuged at 105000 x g for 40 min. The solubilized protein (6-9 mg) was loaded onto a TSK-gel Phenyl-5PW column (7.5 mm I.D. x 7.5 cm, Tosoh) which was connected to a Pharmacia FPLC. Proteins were then eluted with a linear gradient of ammonium sulfate (0.375-0 M) For the hydrolase assay, the enzyme was incubated with 100 /~M [1~4C]anandamide (10000 cpm) at 37°C for 20 min in 200 /ll of 50 m M Tris HC1 H2C-O-CO-RI I
R2-CO-O-CH I
0
0 II
0
AN=V
N-Arachidonyl PE l PLD o
12,~~LOX 12-Hyd roperoxy -
1
~
N
°H
Anandamide m ~ N °~ . . ~
"
Hydrolase COOH
-
anandamide O
(?OH
15-Hydroperoxyanandamide
Arachidonic Acid Fig. 1. Metabolism of anandamide. PE, phosphatidylethanolamine; PLD, phospholipase D; LOX, lipoxygenase.
N. Ueda et al. / J. Lipid Mediators Cell Signalling 14 (1996) 57 61
59
(pH 9.0). For the anandamide synthase assay, the enzyme was incubated with 250 /~M [1-14C]arachidonic acid (50 000 cpm) in 200/~1 of 250 m M ethanolamine-HCl (pH 9.0) at 37°C for 20 min. The products were extracted with diethyl ether, and separated by thin-layer chromatography with the organic phase of a mixture of isooctane/ethyl acetate/water/acetic acid (50:110:100:20, v/v). Distribution of radioactivity on the plate was detected by a Fujix BAS2000 imaging analyzer. Assays were performed in duplicate.
3. Results and discussion
The microsomal fraction of porcine brain was treated with 1% Triton X-100 and the solubilized protein was allowed to react with [~4C]anandamide. When the product was analyzed by thin-layer chromatography, a radioactive band corresponding to arachidonic acid was detected. On the other hand, when the solubilized protein was incubated with [~4C]arachidonic acid, anandamide was produced depending on the presence of ethanolamine. Thus, the solubilized protein from the porcine brain microsome had both the anandamide hydrolase and synthase activities. The solubilized enzyme was loaded onto a Phenyl-5PW hydrophobic column, and each fraction was assayed for the anandamide hydrolase and synthase activities. There were two peaks each showing the two enzyme activities; the first peak containing a bulk of protein and the second peak with the hydrolase purified about 22-fold to a specific enzyme activity of 370 nmol/min per mg protein in a yield of 31%. The anandamide synthase in the second peak was also purified about 21-fold in a yield of 29%, and the specific activity increased to 160 nmol/min per mg protein. The second peak fraction was used as a partially purified enzyme in the following experiments. As the amount of the partially purified enzyme was increased, the hydrolase and synthase activities increased almost in a linear fashion. The hydrolase reaction proceeded linearly up to 30 min while the rate of the anandamide synthase reaction decreased gradually. Substrate specificity of the hydrolase reaction was examined with different fatty acyl ethanolamides. The hydrolase activity increased depending on the concentration of anandamide with an apparent K m of 6 0 / / M . As compared with anandamide at a saturating concentration, the relative hydrolase activity was 44% with linoleylethanolamide, 27% with oleoylethanolamide, and 19% with palmitoylethanolamide. In contrast, the rate of fatty acyl ethanolamide synthesis was not very different with palmitic, oleic, linoleic and arachidonic acids. A very high concentration of ethanolamine (an apparent Km, 50 mM) was required for the full activity of anandamide synthesis. Both the anandamide hydrolase and synthase reactions were active between pH 7 and 9. Furthermore, when the enzyme was preincubated at various temperatures for 5 min, the hydrolase and synthase activities were lost almost in parallel as the temperature was raised. We also tested the effects of various enzyme inhibitors on the anandamide hydrolase and synthase activities. Arachidonyl trifluoromethyl ketone is known as
60
N. Ueda et al. / J. Lipid Mediators Cell Signall&g 14 (1996) 57-61
an inhibitor of cytosolic phospholipase A2, p-chloromercuribenzoic acid as a sulfhydryl blocker, and phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate as serine protease inhibitors. These compounds except PMSF inhibited both the hydrolase and synthase activities almost in parallel depending on their concentrations. A higher concentration of PMSF was required for the inhibition of the synthase activity than the hydrolase activity. Thus, several lines of evidence so far available suggest that both the hydrolase and synthase activities are attributable to a single enzyme protein. This finding must be confirmed by further purification of the enzyme and the cloning and expression of its cDNA. Ceramidase is a well-known lipid-related amidohydrolase, and hydrolyzes ceramide (N-acylsphingosine) to sphingosine and a fatty acid. We examined whether our enzyme preparation was contaminated with the ceramidase which might hydrolyze anandamide. However, the partially purified enzyme showed essentially no ceramidase activity, indicating that the anandamide hydrolase was a separate enzyme from ceramidase. We also tested several artificial substrates for proteases, but all the substrates were inactive with our enzyme. Lipoxygenase enzymes incorporate an oxygen molecule at various positions of polyunsaturated fatty acids, and produce hydroperoxy fatty acids. Since some lipoxygenases are active not only with free unsaturated fatty acids but also with their esters (Yamamoto, 1992), we were interested in whether or not their amides were oxygenated by the lipoxygenases (Ueda et al., 1995). Various lipoxygenases with equivalent activities were incubated with [14C]anandamide, and the products were analyzed by thin-layer chromatography. 5-Lipoxygenase of porcine leukocytes was essentially inactive with anandamide. Purified recombinant human platelet 12-1ipoxygenase produced a faint but significant band of polar product. In contrast, porcine leukocyte 12-1ipoxygenase was active with anandamide. Reticulocyte and soybean 15-1ipoxygenases were also highly active with anandamide. Thus, the lipoxygenases, which have been reported to be capable of oxygenating esterified arachidonic acid of phospholipids (Takahashi et al., 1993) were active with anandamide. For identification of the lipoxygenase products from anandamide, the hydroperoxy products were reduced with sodium borohydride, and analyzed by high-performance liquid chromatography, electron ionization-mass spectrometry, high-resolution FAB mass spectrometry, NMR spectroscopy, and ultraviolet and infrared spectrometry. The major products of 12- and 15-1ipoxygenases were identified as ethanolamides of 12S- and 15S-hydroxyeicosatetraenoic acids, respectively (Fig. 1). It is known that electrically-evoked contraction of murine vas deferens is inhibited by anandamide. We reproduced this inhibitory effect of anandamide with an ICso value of 170 nM. The 15-hydroxy derivative also showed significant activity, while the 12-hydroxy derivative was almost inactive. Thus, the biological activity of anandamide was enzymatically modulated by the lipoxygenase reaction.
N. Ueda et al. / J. Lipid Mediators Cell Signalling 14 (1996) 57-61
61
Acknowledgements We are grateful to Masako Ogawa, Taku Sato, Ichiro Kudo, Keizo Inoue, Hiromasa Takizawa, Tetsuo Nagano, Masaaki Hirobe, Norio Matsuki, and Hiroshi Saito of Faculty of Pharmaceutical Sciences, the University of Tokyo for their collaboration in the bioassay of the oxygenated anandamides. This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, the Human Frontiers in Science Organization, the Japanese Foundation of Metabolism and Disease, and the Japan Research Foundation for Clinical Pharmacology.
References Desarnaud, F., Cadas, H. and Piomelli, D. (1995) Anandamide amidohydrolase activity in rat brain microsomes. Identification and partial characterization. J. Biol. Chem. 270, 6030-6035. Deutsch, D.G. and Chin, S.A. (1993) Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. Biochem. Pharmacol. 46, 791-796. Devane, W.A., Hanus, L., Breuer, A. et al. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946 1949. Devane, W.A. and Axelrod, J. (1994) Enzymatic synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, by brain membranes. Proc. Natl. Acad. Sci. USA 91, 6698 6701. Di Matzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J.-C. and Piomelli, D. (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686-691. Kruszka, K.K. and Gross, R.W. (1994) The ATP- and CoA-independent synthesis of arachidonoylethanolamide. A novel mechanism underlying the synthesis of the endogenous ligand of the cannabinoid receptor. J. Biol. Chem. 269, 14345 14348. Sato, T., Kudo, I. and Inoue, K. (1994) Detection of a phospholipase D hydrolyzing N-arachidonoyl phosphatidylethanolamide. Seikagaku 66, 802 (in Japanese). Takahashi, Y., Glasgow, W.C., Suzuki, H. et al. (1993) Investigation of the oxygenation of phospholipids by the porcine leukocyte and human platelet arachidonate 12-1ipoxygenases. Eur. J. Biochem. 218, 165 171. Ueda, N., Yamamoto, K., Yamamoto, S. et al. (1995) Lipoxygenase-catalyzed oxygenation of arachidonylethanolamide, a cannabinoid receptor agonist. Biochim. Biophys. Acta 1254, 127-134. Yamamoto, S. (1992) Mammalian lipoxygenases: molecular structures and functions. Biochim. Biophys. Acta 1128, 117 131.
LIPID MEDIATORS ANIDC|LL SIGNALLING
ELSEVIER
J. Lipid Mediators Cell Signalling 14 (1996) 57 61
Enzymes for anandamide biosynthesis and metabolism Natsuo Ueda a, Yuko KurahashP, Kei Yamamoto ~, Shozo Yamamoto ~'*, Takashi Tokunaga b aDepartment of Biochemistry, Tokushima University, School of Medicine, Tokushima 770, Japan bJapan Tobacco Inc., Central Pharmaceutical Research Institute, Takatsuki, Osaka 569, Japan
Abstract Anandamide is an endogenous ligand for cannabinoid receptors. We tried to isolate and purify 'anandamide amidohydrolase' which hydrolyzes anandamide to arachidonic acid and ethanolamine. The enzyme activity was found in the microsomal fraction of porcine brain homogenate. The enzyme was solubilized in 1% Triton X-100, and partially purified by hydrophobic chromatography to a specific activity of about 0.3 /~mol/min per mg protein (37°C). Apparent K m for anandamide was about 60 /tM. The enzyme reacted also with ethanolamides of linoleic, oleic, and palmitic acids at lower rates. This enzyme preparation also converted arachidonic acid to anandamide in the presence of 250 mM concentration of ethanolamine. Several lines of evidence including experiments using various inhibitors suggested that the anandamide synthase and amidohydrolase activities were derived from a single enzyme protein. Keywords: Anandamide; Cannabinoid; Lipoxygenase
1. Introduction F o r a better u n d e r s t a n d i n g of the physiological f u n c t i o n of a n a n d a m i d e , a n e n d o g e n o u s ligand for c a n n a b i n o i d receptors ( D e v a n e et al., 1992), we m u s t
* Corresponding author. Tel.: + 81 886 337058; fax: + 81 886 336409. 0929-7855/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0929-7855(96)00509-3
58
N. Ueda et al. / J. Lipid Mediators Cell Signalling 14 (1996) 57 61
elucidate how the production and degradation of this new compound are enzymatically controlled in the cells. As shown in Fig. 1, it was suggested that a phospholipase D was activated and released anandamide from N-arachidonylethanolamide phospholipids (Di Marzo et al., 1994; Sato et al., 1994). The bioactive anandamide was then hydrolyzed and inactivated by a hydrolase (Deutsch and Chin, 1993; Desarnaud et al., 1995). In reverse, anandamide was synthesized by the condensation of arachidonic acid with ethanolamine (Devane and Axelrod, 1994; Kruszka and Gross, 1994). However, the enzymes responsible for these interconversions have not been clearly characterized on the enzymological and molecular biological levels. In this paper we report partial purification and characterization of 'anandamide amidohydrolase' of porcine brain.
2. Materials and methods
The microsomal fraction of porcine brain was suspended in 50 m M Tris-HC1 buffer (pH 8) containing 1% Triton X-100, kept for 12 h, and then centrifuged at 105000 x g for 40 min. The solubilized protein (6-9 mg) was loaded onto a TSK-gel Phenyl-5PW column (7.5 mm I.D. x 7.5 cm, Tosoh) which was connected to a Pharmacia FPLC. Proteins were then eluted with a linear gradient of ammonium sulfate (0.375-0 M) For the hydrolase assay, the enzyme was incubated with 100 /~M [1~4C]anandamide (10000 cpm) at 37°C for 20 min in 200 /ll of 50 m M Tris HC1 H2C-O-CO-RI I
R2-CO-O-CH I
0
0 II
0
AN=V
N-Arachidonyl PE l PLD o
12,~~LOX 12-Hyd roperoxy -
1
~
N
°H
Anandamide m ~ N °~ . . ~
"
Hydrolase COOH
-
anandamide O
(?OH
15-Hydroperoxyanandamide
Arachidonic Acid Fig. 1. Metabolism of anandamide. PE, phosphatidylethanolamine; PLD, phospholipase D; LOX, lipoxygenase.
N. Ueda et al. / J. Lipid Mediators Cell Signalling 14 (1996) 57 61
59
(pH 9.0). For the anandamide synthase assay, the enzyme was incubated with 250 /~M [1-14C]arachidonic acid (50 000 cpm) in 200/~1 of 250 m M ethanolamine-HCl (pH 9.0) at 37°C for 20 min. The products were extracted with diethyl ether, and separated by thin-layer chromatography with the organic phase of a mixture of isooctane/ethyl acetate/water/acetic acid (50:110:100:20, v/v). Distribution of radioactivity on the plate was detected by a Fujix BAS2000 imaging analyzer. Assays were performed in duplicate.
3. Results and discussion
The microsomal fraction of porcine brain was treated with 1% Triton X-100 and the solubilized protein was allowed to react with [~4C]anandamide. When the product was analyzed by thin-layer chromatography, a radioactive band corresponding to arachidonic acid was detected. On the other hand, when the solubilized protein was incubated with [~4C]arachidonic acid, anandamide was produced depending on the presence of ethanolamine. Thus, the solubilized protein from the porcine brain microsome had both the anandamide hydrolase and synthase activities. The solubilized enzyme was loaded onto a Phenyl-5PW hydrophobic column, and each fraction was assayed for the anandamide hydrolase and synthase activities. There were two peaks each showing the two enzyme activities; the first peak containing a bulk of protein and the second peak with the hydrolase purified about 22-fold to a specific enzyme activity of 370 nmol/min per mg protein in a yield of 31%. The anandamide synthase in the second peak was also purified about 21-fold in a yield of 29%, and the specific activity increased to 160 nmol/min per mg protein. The second peak fraction was used as a partially purified enzyme in the following experiments. As the amount of the partially purified enzyme was increased, the hydrolase and synthase activities increased almost in a linear fashion. The hydrolase reaction proceeded linearly up to 30 min while the rate of the anandamide synthase reaction decreased gradually. Substrate specificity of the hydrolase reaction was examined with different fatty acyl ethanolamides. The hydrolase activity increased depending on the concentration of anandamide with an apparent K m of 6 0 / / M . As compared with anandamide at a saturating concentration, the relative hydrolase activity was 44% with linoleylethanolamide, 27% with oleoylethanolamide, and 19% with palmitoylethanolamide. In contrast, the rate of fatty acyl ethanolamide synthesis was not very different with palmitic, oleic, linoleic and arachidonic acids. A very high concentration of ethanolamine (an apparent Km, 50 mM) was required for the full activity of anandamide synthesis. Both the anandamide hydrolase and synthase reactions were active between pH 7 and 9. Furthermore, when the enzyme was preincubated at various temperatures for 5 min, the hydrolase and synthase activities were lost almost in parallel as the temperature was raised. We also tested the effects of various enzyme inhibitors on the anandamide hydrolase and synthase activities. Arachidonyl trifluoromethyl ketone is known as
60
N. Ueda et al. / J. Lipid Mediators Cell Signall&g 14 (1996) 57-61
an inhibitor of cytosolic phospholipase A2, p-chloromercuribenzoic acid as a sulfhydryl blocker, and phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate as serine protease inhibitors. These compounds except PMSF inhibited both the hydrolase and synthase activities almost in parallel depending on their concentrations. A higher concentration of PMSF was required for the inhibition of the synthase activity than the hydrolase activity. Thus, several lines of evidence so far available suggest that both the hydrolase and synthase activities are attributable to a single enzyme protein. This finding must be confirmed by further purification of the enzyme and the cloning and expression of its cDNA. Ceramidase is a well-known lipid-related amidohydrolase, and hydrolyzes ceramide (N-acylsphingosine) to sphingosine and a fatty acid. We examined whether our enzyme preparation was contaminated with the ceramidase which might hydrolyze anandamide. However, the partially purified enzyme showed essentially no ceramidase activity, indicating that the anandamide hydrolase was a separate enzyme from ceramidase. We also tested several artificial substrates for proteases, but all the substrates were inactive with our enzyme. Lipoxygenase enzymes incorporate an oxygen molecule at various positions of polyunsaturated fatty acids, and produce hydroperoxy fatty acids. Since some lipoxygenases are active not only with free unsaturated fatty acids but also with their esters (Yamamoto, 1992), we were interested in whether or not their amides were oxygenated by the lipoxygenases (Ueda et al., 1995). Various lipoxygenases with equivalent activities were incubated with [14C]anandamide, and the products were analyzed by thin-layer chromatography. 5-Lipoxygenase of porcine leukocytes was essentially inactive with anandamide. Purified recombinant human platelet 12-1ipoxygenase produced a faint but significant band of polar product. In contrast, porcine leukocyte 12-1ipoxygenase was active with anandamide. Reticulocyte and soybean 15-1ipoxygenases were also highly active with anandamide. Thus, the lipoxygenases, which have been reported to be capable of oxygenating esterified arachidonic acid of phospholipids (Takahashi et al., 1993) were active with anandamide. For identification of the lipoxygenase products from anandamide, the hydroperoxy products were reduced with sodium borohydride, and analyzed by high-performance liquid chromatography, electron ionization-mass spectrometry, high-resolution FAB mass spectrometry, NMR spectroscopy, and ultraviolet and infrared spectrometry. The major products of 12- and 15-1ipoxygenases were identified as ethanolamides of 12S- and 15S-hydroxyeicosatetraenoic acids, respectively (Fig. 1). It is known that electrically-evoked contraction of murine vas deferens is inhibited by anandamide. We reproduced this inhibitory effect of anandamide with an ICso value of 170 nM. The 15-hydroxy derivative also showed significant activity, while the 12-hydroxy derivative was almost inactive. Thus, the biological activity of anandamide was enzymatically modulated by the lipoxygenase reaction.
N. Ueda et al. / J. Lipid Mediators Cell Signalling 14 (1996) 57-61
61
Acknowledgements We are grateful to Masako Ogawa, Taku Sato, Ichiro Kudo, Keizo Inoue, Hiromasa Takizawa, Tetsuo Nagano, Masaaki Hirobe, Norio Matsuki, and Hiroshi Saito of Faculty of Pharmaceutical Sciences, the University of Tokyo for their collaboration in the bioassay of the oxygenated anandamides. This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, the Human Frontiers in Science Organization, the Japanese Foundation of Metabolism and Disease, and the Japan Research Foundation for Clinical Pharmacology.
References Desarnaud, F., Cadas, H. and Piomelli, D. (1995) Anandamide amidohydrolase activity in rat brain microsomes. Identification and partial characterization. J. Biol. Chem. 270, 6030-6035. Deutsch, D.G. and Chin, S.A. (1993) Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. Biochem. Pharmacol. 46, 791-796. Devane, W.A., Hanus, L., Breuer, A. et al. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946 1949. Devane, W.A. and Axelrod, J. (1994) Enzymatic synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, by brain membranes. Proc. Natl. Acad. Sci. USA 91, 6698 6701. Di Matzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J.-C. and Piomelli, D. (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686-691. Kruszka, K.K. and Gross, R.W. (1994) The ATP- and CoA-independent synthesis of arachidonoylethanolamide. A novel mechanism underlying the synthesis of the endogenous ligand of the cannabinoid receptor. J. Biol. Chem. 269, 14345 14348. Sato, T., Kudo, I. and Inoue, K. (1994) Detection of a phospholipase D hydrolyzing N-arachidonoyl phosphatidylethanolamide. Seikagaku 66, 802 (in Japanese). Takahashi, Y., Glasgow, W.C., Suzuki, H. et al. (1993) Investigation of the oxygenation of phospholipids by the porcine leukocyte and human platelet arachidonate 12-1ipoxygenases. Eur. J. Biochem. 218, 165 171. Ueda, N., Yamamoto, K., Yamamoto, S. et al. (1995) Lipoxygenase-catalyzed oxygenation of arachidonylethanolamide, a cannabinoid receptor agonist. Biochim. Biophys. Acta 1254, 127-134. Yamamoto, S. (1992) Mammalian lipoxygenases: molecular structures and functions. Biochim. Biophys. Acta 1128, 117 131.