Journal of Endocrinology and Reproduction

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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
     

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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.
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  • Purification and Characterization of LTC4 Synthase from Sheep Uterus

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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