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. 1997 Oct 1;327(Pt 1):193–198. doi: 10.1042/bj3270193

Characterization and purification of a lipoxygenase inhibitor in human epidermoid carcinoma A431 cells.

C J Chen 1, H S Huang 1, Y T Lee 1, C Y Yang 1, W C Chang 1
PMCID: PMC1218780  PMID: 9355752

Abstract

A lipoxygenase inhibitor in the cytosolic fraction of human epidermoid carcinoma A431 cells was characterized and purified. The cytosolic inhibitor lost the inhibitory activity upon heating at 75 degrees C for 15 min or pretreating with 1 mg/ml trypsin at 37 degrees C for 60 min. Cytosol, after dialysis, lost the inhibitory activity but its inhibitory activity recovered when 1 mM GSH was added to the dialysate. The inhibitory activity of cytosol was also abolished by treatment either with 1 mM iodoacetate at 4 degrees C for 1 h or with 0.5 mM H2O2. The pI of the inhibitor was approx. 7.0. In addition to 12-lipoxygenase, the inhibitor inhibited the activities of 5-lipoxygenase and fatty acid cyclo-oxygenase in a cell-free system. The inhibitor was purified by a series of column chromatographies using CM Sephadex C-50, Sephadex G-100 SF and Mono P columns. A major 22 kDa protein was obtained that was distinct from selenium-dependent glutathione peroxidase.

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Selected References

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  1. Burk R. F., Trumble M. J., Lawrence R. A. Rat hepatic cytosolic glutathione-dependent enzyme protection against lipid peroxidation in the NADPH-microsomal lipid peroxidation system. Biochim Biophys Acta. 1980 Apr 18;618(1):35–41. doi: 10.1016/0005-2760(80)90051-x. [DOI] [PubMed] [Google Scholar]
  2. Chan H. W. Soya-bean lipoxygenase: an iron-containing dioxygenase. Biochim Biophys Acta. 1973 Nov 15;327(1):32–35. doi: 10.1016/0005-2744(73)90100-9. [DOI] [PubMed] [Google Scholar]
  3. Chang W. C., Liu Y. W., Ning C. C., Suzuki H., Yoshimoto T., Yamamoto S. Induction of arachidonate 12-lipoxygenase mRNA by epidermal growth factor in A431 cells. J Biol Chem. 1993 Sep 5;268(25):18734–18739. [PubMed] [Google Scholar]
  4. Chang W. C., Nakao J., Orimo H., Murota S. Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets. Biochem J. 1982 Mar 15;202(3):771–776. doi: 10.1042/bj2020771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang W. C., Ning C. C., Lin M. T., Huang J. D. Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. J Biol Chem. 1992 Feb 25;267(6):3657–3666. [PubMed] [Google Scholar]
  6. Chen Y. Q., Duniec Z. M., Liu B., Hagmann W., Gao X., Shimoji K., Marnett L. J., Johnson C. R., Honn K. V. Endogenous 12(S)-HETE production by tumor cells and its role in metastasis. Cancer Res. 1994 Mar 15;54(6):1574–1579. [PubMed] [Google Scholar]
  7. Gibson D. D., Hawrylko J., McCay P. B. GSH-dependent inhibition of lipid peroxidation: properties of a potent cytosolic system which protects cell membranes. Lipids. 1985 Oct;20(10):704–711. doi: 10.1007/BF02534391. [DOI] [PubMed] [Google Scholar]
  8. Gibson D. D., Hornbrook K. R., McCay P. B. Glutathione-dependent inhibition of lipid peroxidation by a soluble, heat-labile factor in animal tissues. Biochim Biophys Acta. 1980 Dec 5;620(3):572–582. doi: 10.1016/0005-2760(80)90149-6. [DOI] [PubMed] [Google Scholar]
  9. Kamataki T., Ozawa N., Kitada M., Kitagawa H., Sato R. The occurrence of an inhibitor of lipid peroxidation in rat liver soluble fraction and its effect on microsomal drug oxidations. Biochem Pharmacol. 1974 Sep 1;23(17):2485–2490. doi: 10.1016/0006-2952(74)90248-2. [DOI] [PubMed] [Google Scholar]
  10. Karthein R., Dietz R., Nastainczyk W., Ruf H. H. Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction. Eur J Biochem. 1988 Jan 15;171(1-2):313–320. doi: 10.1111/j.1432-1033.1988.tb13792.x. [DOI] [PubMed] [Google Scholar]
  11. Kotake A. N., Deloria L. B., Abbott V. S., Mannering G. J. Reversal of the inhibitory effect of lipid peroxides on the hepatic cytochrome P-450 monooxygenase system by a soluble factor from liver and a commercial isocitric dehydrogenase preparation from hog heart. Biochem Biophys Res Commun. 1975 Mar 3;63(1):209–216. doi: 10.1016/s0006-291x(75)80031-3. [DOI] [PubMed] [Google Scholar]
  12. Kujubu D. A., Reddy S. T., Fletcher B. S., Herschman H. R. Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells. J Biol Chem. 1993 Mar 15;268(8):5425–5430. [PubMed] [Google Scholar]
  13. Kulmacz R. J., Tsai A. L., Palmer G. Heme spin states and peroxide-induced radical species in prostaglandin H synthase. J Biol Chem. 1987 Aug 5;262(22):10524–10531. [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Liu Y. W., Chen B. K., Chen C. J., Arakawa T., Yoshimoto T., Yamamoto S., Chang W. C. Epidermal growth factor enhances transcription of human arachidonate 12-lipoxygenase in A431 cells. Biochim Biophys Acta. 1997 Jan 7;1344(1):38–46. doi: 10.1016/s0005-2760(96)00128-2. [DOI] [PubMed] [Google Scholar]
  17. Matsuda S., Suzuki H., Yoshimoto T., Yamamoto S., Miyatake A. Analysis of non-heme iron in arachidonate 12-lipoxygenase of porcine leukocytes. Biochim Biophys Acta. 1991 Jul 9;1084(2):202–204. [PubMed] [Google Scholar]
  18. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  19. Morita I., Schindler M., Regier M. K., Otto J. C., Hori T., DeWitt D. L., Smith W. L. Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem. 1995 May 5;270(18):10902–10908. doi: 10.1074/jbc.270.18.10902. [DOI] [PubMed] [Google Scholar]
  20. Nakamura M., Ueda N., Kishimoto K., Yoshimoto T., Yamamoto S., Ishimura K. Immunocytochemical localization of platelet-type arachidonate 12-lipoxygenase in mouse blood cells. J Histochem Cytochem. 1995 Mar;43(3):237–244. doi: 10.1177/43.3.7868854. [DOI] [PubMed] [Google Scholar]
  21. Nelson D. O., Lorusso D. J., Mannering G. J. Requirement of a soluble protein for maximal activity of the mono-oxidase system of hepatic microsomes. Biochem Biophys Res Commun. 1973 Aug 6;53(3):995–1001. doi: 10.1016/0006-291x(73)90190-3. [DOI] [PubMed] [Google Scholar]
  22. Otto J. C., Smith W. L. The orientation of prostaglandin endoperoxide synthases-1 and -2 in the endoplasmic reticulum. J Biol Chem. 1994 Aug 5;269(31):19868–19875. [PubMed] [Google Scholar]
  23. Percival M. D. Human 5-lipoxygenase contains an essential iron. J Biol Chem. 1991 Jun 5;266(16):10058–10061. [PubMed] [Google Scholar]
  24. Powell S. R., McCay P. B. Inhibition of doxorubicin-initiated membrane damage by N-acetylcysteine: possible mediation by a thiol-dependent, cytosolic inhibitor of lipid peroxidation. Toxicol Appl Pharmacol. 1988 Nov;96(2):175–184. doi: 10.1016/0041-008x(88)90078-6. [DOI] [PubMed] [Google Scholar]
  25. Reiger M. K., DeWitt D. L., Schindler M. S., Smith W. L. Subcellular localization of prostaglandin endoperoxide synthase-2 in murine 3T3 cells. Arch Biochem Biophys. 1993 Mar;301(2):439–444. doi: 10.1006/abbi.1993.1168. [DOI] [PubMed] [Google Scholar]
  26. Schewe T., Rapoport S. M., Kühn H. Enzymology and physiology of reticulocyte lipoxygenase: comparison with other lipoxygenases. Adv Enzymol Relat Areas Mol Biol. 1986;58:191–272. doi: 10.1002/9780470123041.ch6. [DOI] [PubMed] [Google Scholar]
  27. Shornick L. P., Holtzman M. J. A cryptic, microsomal-type arachidonate 12-lipoxygenase is tonically inactivated by oxidation-reduction conditions in cultured epithelial cells. J Biol Chem. 1993 Jan 5;268(1):371–376. [PubMed] [Google Scholar]
  28. Smith W. L., Marnett L. J. Prostaglandin endoperoxide synthase: structure and catalysis. Biochim Biophys Acta. 1991 Apr 24;1083(1):1–17. doi: 10.1016/0005-2760(91)90119-3. [DOI] [PubMed] [Google Scholar]
  29. Yamamoto S. "Enzymatic" lipid peroxidation: reactions of mammalian lipoxygenases. Free Radic Biol Med. 1991;10(2):149–159. doi: 10.1016/0891-5849(91)90008-q. [DOI] [PubMed] [Google Scholar]
  30. de Groot J. J., Veldink G. A., Vliegenthart J. F., Boldingh J., Wever R., van Gelder B. F. Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1. Biochim Biophys Acta. 1975 Jan 23;377(1):71–79. doi: 10.1016/0005-2744(75)90287-9. [DOI] [PubMed] [Google Scholar]

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