Abstract
Leukotriene A4 synthase was purified from the cytosolic fraction of murine mast cells. The enzyme converted 5-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HPETE) to leukotriene A4. This unstable product was identified by demonstration of two epimers of 6-transleukotriene B4, methanol trapping, as well as further transformation to leukotriene B4 by leukotriene A4 hydrolase. Leukotriene A4 synthase stereospecifically eliminated the D-hydrogen at C-10 (pro-R) in the synthesis of leukotriene A4 when incubated with [10D-3H;3-(14)C]5-HPETE. The purified enzyme also exhibited 5-lipoxygenase activity toward arachidonic acid and 8-lipoxygenase activity towards 8,11,14-cisicosatrienoic acid. All of these activities required Ca2+ and ATP for their maximal velocities. The effects of heat treatment and of several lipoxygenase inhibitors on these enzyme activities as well as coelution in various chromatographic systems strongly suggest that lipoxygenase and leukotriene A4 synthase activities reside in the same enzyme molecule.
Full text
PDF![4175](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e88e/323694/e1e26fcbf81a/pnas00316-0075.png)
![4176](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e88e/323694/8cc16cec58ed/pnas00316-0076.png)
![4177](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e88e/323694/a8a19c97e5c6/pnas00316-0077.png)
![4178](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e88e/323694/17903c9e36ab/pnas00316-0078.png)
![4179](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e88e/323694/1a19debeda71/pnas00316-0079.png)
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ball P. E., Conroy M. C., Heusser C. H., Davis J. M., Conscience J. F. Spontaneous, in vitro, malignant transformation of a basophil/mast cell line. Differentiation. 1983;24(1):74–78. doi: 10.1111/j.1432-0436.1983.tb01305.x. [DOI] [PubMed] [Google Scholar]
- Borgeat P., Samuelsson B. Arachidonic acid metabolism in polymorphonuclear leukocytes: unstable intermediate in formation of dihydroxy acids. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3213–3217. doi: 10.1073/pnas.76.7.3213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Goetze A. M., Fayer L., Bouska J., Bornemeier D., Carter G. W. Purification of a mammalian 5-lipoxygenase from rat basophilic leukemia cells. Prostaglandins. 1985 May;29(5):689–701. doi: 10.1016/0090-6980(85)90130-3. [DOI] [PubMed] [Google Scholar]
- Jakschik B. A., Kuo C. G. Characterization of leukotriene A4 and B4 biosynthesis. Prostaglandins. 1983 Jun;25(6):767–782. doi: 10.1016/0090-6980(83)90002-3. [DOI] [PubMed] [Google Scholar]
- Jakschik B. A., Lee L. H. Enzymatic assembly of slow reacting substance. Nature. 1980 Sep 4;287(5777):51–52. doi: 10.1038/287051a0. [DOI] [PubMed] [Google Scholar]
- Maas R. L., Ingram C. D., Taber D. F., Oates J. A., Brash A. R. Stereospecific removal of the DR hydrogen atom at the 10-carbon of arachidonic acid in the biosynthesis of leukotriene A4 by human leukocytes. J Biol Chem. 1982 Nov 25;257(22):13515–13519. [PubMed] [Google Scholar]
- Ochi K., Yoshimoto T., Yamamoto S., Taniguchi K., Miyamoto T. Arachidonate 5-lipoxygenase of guinea pig peritoneal polymorphonuclear leukocytes. Activation by adenosine 5'-triphosphate. J Biol Chem. 1983 May 10;258(9):5754–5758. [PubMed] [Google Scholar]
- Panossian A., Hamberg M., Samuelsson B. On the mechanism of biosynthesis of leukotrienes and related compounds. FEBS Lett. 1982 Dec 27;150(2):511–513. doi: 10.1016/0014-5793(82)80801-6. [DOI] [PubMed] [Google Scholar]
- Rouzer C. A., Matsumoto T., Samuelsson B. Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities. Proc Natl Acad Sci U S A. 1986 Feb;83(4):857–861. doi: 10.1073/pnas.83.4.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rouzer C. A., Samuelsson B. On the nature of the 5-lipoxygenase reaction in human leukocytes: enzyme purification and requirement for multiple stimulatory factors. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6040–6044. doi: 10.1073/pnas.82.18.6040. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rouzer C. A., Shimizu T., Samuelsson B. On the nature of the 5-lipoxygenase reaction in human leukocytes: characterization of a membrane-associated stimulatory factor. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7505–7509. doi: 10.1073/pnas.82.22.7505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rådmark O., Shimizu T., Fitzpatrick F., Samuelsson B. Hemoprotein catalysis of leukotriene formation. Biochim Biophys Acta. 1984 Mar 7;792(3):324–329. doi: 10.1016/0005-2760(84)90200-5. [DOI] [PubMed] [Google Scholar]
- Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science. 1983 May 6;220(4597):568–575. doi: 10.1126/science.6301011. [DOI] [PubMed] [Google Scholar]
- Soberman R. J., Harper T. W., Betteridge D., Lewis R. A., Austen K. F. Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes. J Biol Chem. 1985 Apr 10;260(7):4508–4515. [PubMed] [Google Scholar]
- Warner N. L., Moore M. A., Metcalf D. A transplantable myelomonocytic leukemia in BALB-c mice: cytology, karyotype, and muramidase content. J Natl Cancer Inst. 1969 Oct;43(4):963–982. [PubMed] [Google Scholar]
- Yoshimoto T., Yokoyama C., Ochi K., Yamamoto S., Maki Y., Ashida Y., Terao S., Shiraishi M. 2,3,5-Trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of the 5-lipoxygenase reaction and the biosynthesis of slow-reacting substance of anaphylaxis. Biochim Biophys Acta. 1982 Nov 12;713(2):470–473. [PubMed] [Google Scholar]