Journal of Endocrinology and Reproduction

Open Access Open Access  Restricted Access Subscription Access
Open Access Open Access Open Access  Restricted Access Restricted Access Subscription Access

Purification and Characterization of LTC4 Synthase from Sheep Uterus


Affiliations
1 Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, Telangana, India
2 Department of Biotechnology and Bioinformatics, Yogi Vemana University, Kadapa – 516003, Andhra Pradesh, India
     

   Subscribe/Renew Journal


Purification and Characterization of LTC4 Synthase from Sheep UterusPurification and Characterization of LTC4 Synthase from Sheep UterusEicosanoids, the oxygenated metabolites of eicosapolyenoic fatty acids such as arachidonic acid via the cyclooxygenase (COX), lipoxygenase (LOX) and epoxygenase (EPOX) pathways, are generated in response to specific stimuli, elicit the response and are then quickly metabolized. Hence, these are rightly termed as “local hormones” or “autocoids”. They are involved in the regulation of a variety of physiological as well as pathological processes, including reproduction. While there are extensive studies on the role of COX metabolites, such as prostaglandins, in reproduction, not much is known on the role of LOX metabolites in reproduction. Earlier, we have identified abundant LOX activity in sheep uterus and the highly purified enzyme was found to be a homo-dimeric protein with a molecular weight of 66 kDa. When incubated with arachidonic acid, the enzyme showed two lipoxygenase activities producing both 12- and 15-Hydroxyeicosatetraenoic acid (15-HPETEs) at the optimum pH of 5.5. The relative concentration of 12- and 15-HPETEs, however, changed with the pH of the reaction, 12-Hydroxyeicosatetraenoic acid (HETE) being higher in the alkaline range and 15-HETE being the abundant in the acidic range. Furthermore, the enzyme showed the dual lipoxygenase based 14,15-LTA4 synthase activity as evidenced by the formation of 8,15-diHETEs, the hydrolysis products of 14,15-LTA4. In the present study, leukotriene C4 synthase (LTC4S) enzyme was purified on Q-Sepharose column after solubilization of microsomes utilizing a combination of CHAPS and taurocholate. The purified enzyme showed activity with 5, 6-LTA4 and 14, 15-LTA4, with slight preference towards the latter, and converting them to corresponding LTC4s. Both methyl esters and free acids of LTA4 served as substrates, though the activity was more with methyl esters. However, the enzyme showed no activity with I-chloro-2, 4-dinitrobenzene (CDNB), the conventional substrate of glutathione S-transferases. Western blot analysis of sheep uterine microsomal proteins with LTC4 synthase specific-peptide as well as whole protein antibodies showed strong cross reactivity with two closely migrating 70 kDa proteins. While showing similarity with the known LTC4 synthases, sheep uterine LTC4 synthase thus appears to be quite different in terms of molecular weight, as most LTC4 synthases reported are 18 kDa proteins. In view of its association with the microsomal membranes and involvement in eicosanoid and glutathione metabolism, sheep uterine LTC4 synthase may form a member of MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily.

Keywords

Eicosanoids, Lipoxygenase, Sheep Uterine LTC4 Synthase, MAPEG Superfamily.
Subscription Login to verify subscription
User
Notifications
Font Size


  • Wang D, DuBois RN. Eicosanoids and cancer. Nature Reviews Cancer. 2010; 10(3):181–93. https://doi.org/10.1038/nrc2809 PMid:20168319 PMCid:PMC2898136

  • Qiu H, Gabrielsen A, Agardh HE, et al. Expression of 5-lipoxygenase and leukotriene A4 hydrolase in human atherosclerotic lesions correlates with symptoms of plaque instability. Proceedings of the National Academy of Sciences. 2006; 103(21):8161–6. https://doi.org/10.1073/pnas.0602414103 PMid:16698924 PMCid:PMC1459628

  • Shao WH, Del Prete A, Bock CB, Haribabu B. Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice. The Journal of Immunology. 2006; 176(10):6254–61. https://doi.org/10.4049/jimmunol.176.10.6254 PMid:16670336

  • Jana B, Czarzasta J, Jaroszewski J. Synthesis of leukotrienes in porcine uteri with endometritis induced by infection with Escherichia coli. Reproduction, Fertility and Development. 2014; 26(7):1007–16. https://doi.org/10.1071/RD13191 PMid:23924575

  • Hallstrand TS, Henderson Jr WR. An update on the role of leukotrienes in asthma. Current Opinion in Allergy and Clinical Immunology. 2010; 10(1):60. https://doi. org/10.1097/ACI.0b013e32833489c3 PMid:19915456 PMCid:PMC2838730

  • Liu M, Yokomizo T. The role of leukotrienes in allergic diseases. Allergology International. 2015; 64(1):17–26. https://doi.org/10.1016/j.alit.2014.09.001 PMid:25572555

  • Behl T, Kaur I, Kotwani A. Role of leukotrienes in diabetic retinopathy. Prostaglandins &Other Lipid Mediators. 2016; 122:1–9. https://doi.org/10.1016/j.prostaglandins.2015.12.001 PMid:26673555

  • RouzerC, Kargman S. Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187. The Journal of Biological Chemistry. 1988; 263(22):10980–8. PMid:3134355

  • Chang M, Rao MK, Reddanna P, Li CH, Tu CP, Corey EJ, Reddy CC. I. Specificity of the glutathione S-transferases in the conversion of leukotriene A4 to leukotriene C4. Archives of Biochemistry and Biophysics. 1987; 259(2):536–47. https://doi.org/10.1016/0003-9861%2887%2990520-0

  • Izumi T, Honda Z, Ohishi N, et al. Solubilization and partial purification of leukotriene C4 synthase from guinea-pig lung: a microsomal enzyme with high specificity towards 5,6-epoxide leukotriene A4. BiochimicaetBiophysicaActa. 1988; 959(3):305–15.

  • Jakobsson PJ, Morgenstern R, Mancini J, Ford‐Hutchinson A, Persson B. Common structural features of MAPEG—a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism. Protein Science. 1999;8(3): 689–92. https://doi.org/10.1110/ps.8.3.689 PMid:10091672 PMCid:PMC2144274

  • Weller PF, Lee CW, Foster DW, Corey EJ, Austen KF, Lewis RA. Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4. Proceedings of the National Academy of Sciences USA. 1983; 80(24):7626–30. https://doi.org/10.1073/pnas.80.24.7626

  • Williams JD, Czop JK, Austen KF. Release of leukotrienes by human monocytes on stimulation of their phagocytic receptor for particulate activators. Journal of Immunology. 1984; 132(6):3034–40. PMid:6327815

  • Maclouf JA, Murphy RC. Transcellular metabolism of neutrophil-derived leukotriene A4 by human platelets.A potential cellular source of leukotriene C4. Journal of Biological Chemistry. 1988; 263(1):174–81. PMid:2826437

  • Penrose JF, Gagnon L, Goppelt-StruebeM,et al. Purification of human leukotriene C4 synthase. Proceedings of the National Academy of Sciences USA. 1992; 89(23):11603–6. https://doi.org/10.1073/pnas.89.23.11603

  • Nicholson DW, Ali AM, Klemba MW, Munday NA, Zamboni RJ, Ford-Hutchinson AW. Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells. Journal of Biological Chemistry. 1992; 267(25):17849–57. PMid:1517222

  • Nicholson DW, Ali A, Vaillancourt JP, Calaycay JR, Mumford RA, Zamboni RJ, Ford-Hutchinson AW. Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits.Proceedings of the National Academy of Sciences USA. 1993; 90(5):2015–9. https://doi.org/10.1073/pnas.90.5.2015

  • Ali A, Zamboni RJ, Ford-Hutchinson AW, Nicholson DW. Photoaffinity labeling of human leukotriene C4 synthase in THP-1 cell membranes. FEBS Letters. 1993; 317(3):195–201. https://doi.org/10.1016/0014-5793%2893%2981275-5

  • Feinmark SJ, Cannon PJ. Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes. Journal of Biological Chemistry. 1986; 261(35):16466–72. PMid:3023351

  • Penrose JF, Spector J, Lam BK, Friend DS, Xu K, Jack RM, Austen KF. Purification of human lung leukotriene C4 synthase and preparation of a polyclonal antibody. American Journal of Respiratory and Critical Care Medicine. 1995; 152(1):283–9. https://doi.org/10.1164/ajrccm.152.1.7599836 PMid:7599836

  • Petric R, Nicholson DW, Ford-Hutchinson AW. Renal leukotriene C4 synthase: characterization, partial purification and alterations in experimental glomerulonephritis. Biochimica et Biophysica Acta. (BBA)- Lipids and Lipid Metabolism. 1995; 1254(2):207–15. https://doi.org/10.1016/0005-2760%2894%2900185-2

  • Nicholson DW, Ali AM, Klemba MW, Munday NA, Zamboni RJ, Ford-Hutchinson AW. Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells. Journal of Biological Chemistry. 1992; 267(25):17849–57. PMid:1517222

  • Reddanna P, Whelan JK, Maddipati KR, Reddy CC. Purification of arachidonate 5-lipoxygenase from potato tubers. Methods in Enzymology. 1990; 187:268–77. Crossref

  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA. 1979; 76(9):4350–4. https://doi.org/10.1073/pnas.76.9.4350

  • Wu C. Conversion of leukotrienes A4 to C4 in cellfree systems. Biochemical and Biophysical Research

  • Communication. 1986; 134(1):85–92. https://doi.org/10.1016/0006-291X%2886%2990530-9

  • Nicholson DW, Klemba MW, Rasper DM, Metters KM, Zamboni RJ, Ford‐Hutchinson AW. Purification of human leukotriene C4 synthase from dimethylsulfoxide‐differentiated U937 cells. The FEBS Journal. 1992; 209(2):725–34. https://doi.org/10.1111/j.1432-1033.1992.tb17341.x

  • Abe M, Hara N, Muranishi H, Ikeda T, Nagata N, Shigematsu N. Enhanced leukotriene C4 synthase activity in thioglycollateelicited peritoneal macrophages. Biochemical and Biophysical Research Communications. 1990; 171(3):1344–52. https://doi.org/10.1016/0006-291X%2890%2990834-A

  • Yoshimoto T, Soberman RJ, Spur B, Austen KF. Properties of highly purified leukotriene C4 synthase of guinea pig lung. Journal of Clinical Investigation. 1988; 81(3):866–71. https://doi.org/10.1172/JCI113396 PMid:3343345 PMCid:PMC442538

  • Ali A, Zamboni RJ, Ford-Hutchinson AW, Nicholson DW. Photoaffinity labelling of human leukotriene C4 synthase in THP-1 cell membranes. FEBS Letters. 1993; 317(3):195–201. https://doi.org/10.1016/0014-5793%2893%2981275-5

  • Sailesh S, Kumar YK, Prasad M, Reddanna P. Sheep uterus dual lipoxygenase in the synthesis of 14, 15-leukotrienes. Archives of Biochemistry and Biophysics. 1994; 315(2):362–8. https://doi.org/10.1006/abbi.1994.1512 PMid:7986079

  • Nicholson DW, Ali A, Vaillancourt JP, Calaycay JR, Mumford RA, Zamboni RJ, Ford-Hutchinson AW. Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits. The Proceedings of the National Academy of Sciences U S A. 1993; 90(5):2015–9. https://doi.org/10.1073/pnas.90.5.2015

  • Jenny Y. Purification and characterization of glutathione S-transferases from rat

  • Jakobsson PJ, Mancini JA, Riendeau D, Ford-Hutchinson AW. Identification and characterization of a novel microsomal enzyme with glutathione-dependent transferase and peroxidase activities. Journal of Biological Chemistry. 1997; 272(36):22934–9. https://doi.org/10.1074/jbc.272.36.22934 PMid:9278457

  • Akobsson PJ, Mancini JA, Ford-Hutchinson AW.Identification and characterization of a novel human microsomal glutathione S-transferase with leukotriene C4 synthase activity and significant sequence identity to 5-lipoxygenase-activating protein and leukotriene C4 synthase. Journal of Biological Chemistry. 1996; 271(36):22203–10. https://doi.org/10.1074/jbc.271.36.22203%20 PMid:8703034


Abstract Views: 340

PDF Views: 1




  • Purification and Characterization of LTC4 Synthase from Sheep Uterus

Abstract Views: 340  |  PDF Views: 1

Authors

B. Muralidhar Reddy
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, Telangana, India
Madhava C. Reddy
Department of Biotechnology and Bioinformatics, Yogi Vemana University, Kadapa – 516003, Andhra Pradesh, India
Vengala Rao Yenuganti
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, Telangana, India
Gorla V. Reddy
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, Telangana, India
Pallu Reddanna
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, Telangana, India

Abstract


Purification and Characterization of LTC4 Synthase from Sheep UterusPurification and Characterization of LTC4 Synthase from Sheep UterusEicosanoids, the oxygenated metabolites of eicosapolyenoic fatty acids such as arachidonic acid via the cyclooxygenase (COX), lipoxygenase (LOX) and epoxygenase (EPOX) pathways, are generated in response to specific stimuli, elicit the response and are then quickly metabolized. Hence, these are rightly termed as “local hormones” or “autocoids”. They are involved in the regulation of a variety of physiological as well as pathological processes, including reproduction. While there are extensive studies on the role of COX metabolites, such as prostaglandins, in reproduction, not much is known on the role of LOX metabolites in reproduction. Earlier, we have identified abundant LOX activity in sheep uterus and the highly purified enzyme was found to be a homo-dimeric protein with a molecular weight of 66 kDa. When incubated with arachidonic acid, the enzyme showed two lipoxygenase activities producing both 12- and 15-Hydroxyeicosatetraenoic acid (15-HPETEs) at the optimum pH of 5.5. The relative concentration of 12- and 15-HPETEs, however, changed with the pH of the reaction, 12-Hydroxyeicosatetraenoic acid (HETE) being higher in the alkaline range and 15-HETE being the abundant in the acidic range. Furthermore, the enzyme showed the dual lipoxygenase based 14,15-LTA4 synthase activity as evidenced by the formation of 8,15-diHETEs, the hydrolysis products of 14,15-LTA4. In the present study, leukotriene C4 synthase (LTC4S) enzyme was purified on Q-Sepharose column after solubilization of microsomes utilizing a combination of CHAPS and taurocholate. The purified enzyme showed activity with 5, 6-LTA4 and 14, 15-LTA4, with slight preference towards the latter, and converting them to corresponding LTC4s. Both methyl esters and free acids of LTA4 served as substrates, though the activity was more with methyl esters. However, the enzyme showed no activity with I-chloro-2, 4-dinitrobenzene (CDNB), the conventional substrate of glutathione S-transferases. Western blot analysis of sheep uterine microsomal proteins with LTC4 synthase specific-peptide as well as whole protein antibodies showed strong cross reactivity with two closely migrating 70 kDa proteins. While showing similarity with the known LTC4 synthases, sheep uterine LTC4 synthase thus appears to be quite different in terms of molecular weight, as most LTC4 synthases reported are 18 kDa proteins. In view of its association with the microsomal membranes and involvement in eicosanoid and glutathione metabolism, sheep uterine LTC4 synthase may form a member of MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily.

Keywords


Eicosanoids, Lipoxygenase, Sheep Uterine LTC4 Synthase, MAPEG Superfamily.

References





DOI: https://doi.org/10.18311/jer%2F2017%2F21065