In Vitro Effects of Diclofenac Sodium on Glucose-6-Phosphate Dehydrogenase and Glutathione Reductase Enzymes Purified from Sheep Spleen Tissue

Öz

In this study, in vitro effects of diclofenac sodium, which is used to prevent inflammation and alleviate pain in arthritis and acute injuries, on glucose-6-phosphate dehydrogenase enzyme (G6PD; EC 1.1.1.49) and glutathione reductase enzyme (GR; EC 1.8.1.7) purified from sheep spleen tissue were investigated. In the first stage of the study, G6PD and GR enzymes were purified from sheep spleen tissue by ammonium sulfate (NH4)2SO4 precipitation and 2', 5' ADP-Sepharose-4B gel affinity chromatography. The purity levels of the enzymes were checked by the sodyum dodesilsülfat poliakrilamid jel elektroforezi (SDS-PAGE) method. In the second stage of the study, the effects of diclofenac sodium on the pure enzyme activities were investigated. IC50 values were calculated by plotting the % Activity-[I] graph for diclofenac sodium, which has an inhibitory effect on enzymes. In addition, Lineweaver-Burk graphs were drawn by making measurements with five different substrate concentrations and three different inhibitor concentrations. From these graphs, it was determined that G6PD enzyme inhibited with 0.51 mM IC50 value and 0.48 ± 0.01 mM Ki value non competitively, by diclofenac sodium. It was determined that the GR enzyme inhibited with an 6.55 mM IC50 value of and 15.69 ± 0.84 mM Ki value non competitively.

Anahtar Kelimeler

• G6PD
• GR
• diclofenac sodium
• inhibition

Koyun Dalak Dokusundan Saflaştırılan Glukoz-6-Fosfat Dehidrogenaz ve Glutatyon Redüktaz Enzimi Üzerine Diklofenak Sodyumun İn Vitro Etkileri

Öz

Bu çalışmada, artrit ve akut yaralanmalarda enflamasyonu önlemek ve ağrıyı dindirmek için kullanılan diklofenak sodyumun, koyun dalak dokusundan saflaştırılan glukoz-6-fosfat dehidrogenaz enzimi (G6PD; E.C. 1.1.1.49) ve glutatyon redüktaz enzimi (GR; E.C. 1.8.1.7) üzerine in vitro etkileri araştırıldı. Çalışmanın ilk aşamasında G6PD ve GR enzimleri koyun dalak dokusundan, amonyum sülfat (NH4)2SO4 çöktürmesi ve 2', 5' ADP-Sepharose-4B jel afinite kromatografisi ile saflaştırıldı. Enzimlerin saflık dereceleri sodyum dodesilsülfat poliakrilamid jel elektroforezi (SDS-PAGE) metodu ile kontrol edildi. Çalışmanın ikinci aşamasında saf olarak elde edilen enzim aktiviteleri üzerine diklofenak sodyumun etkileri araştırıldı. Enzimler üzerinde inhibisyon etkisi gösteren diklofenak sodyumun için % Aktivite-[I] grafiği çizilerek IC50 değerleri hesaplandı. Ayrıca beş farklı substrat konsantrasyonu ve üç farklı inhibitör konsantrasyonlarıyla ölçümler yapılarak Lineweaver-Burk grafikleri çizildi. Elde edilen bu grafiklerden diklofenak sodyumun G6PD enzimini 0.51 mM, IC50 değeri ve 0.48 ± 0.01 mM Ki değeri ile yarışmasız olarak inhibe ettiği belirlendi. GR enzimini ise 6.55 mM IC50 değeri ve 15.69 ± 0.84 mM Ki değeri ile yarışmasız olarak inhibe ettiği tespit edildi.

Anahtar Kelimeler

• G6PD
• GR
• diklofenak sodyum
• inhibisyon

Kaynakça

1. Adem S, Ciftci M, 2012. Purification of rat kidney glucose 6 phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2ˈ, 5ˈ-ADP sepharose 4B affinity in a single chromatography step. Protein Expression and Purification, 81(1):1-4.
2. Adem S, Ciftci M, 2016. Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics. Journal of enzyme inhibition and medicinal chemistry, 31(6); 1342-1348.
3. Akkemik E, Şentürk M, Özgeriş FB, Taşer P, Çiftci M, 2011. In vitro effects of some drugs on human erythrocyte glutathione reductase, Turkish Journal of Medical Sciences, 41(2): 235-41.
4. Bayramoğlu Akkoyun M, Bengü AŞ, Temel Y, Akkoyun HT, Ekin S, Ciftci M, 2018. The effect of astaxanthin and cadmium on rat erythrocyte G6PD, 6PGD, GR, and TrxR enzymes activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro. Journal of biochemical and molecular toxicology, 32(8), e22170.
5. Beutler E, 1971. Red Cell Metabolism Manual of Biochemical Methods; Academic Press: London,
6. Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analitical Biochemistry, 72(12): 248-254. Carlberg I, Mannervik B, 1981. Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2’, 5’-ADP-Sepharose 4B. Analytical Biochemistry, (116):531-536.
7. Ciftci M, Kufrevioglu OI, Gundogdu M, Ozmen I, 2000. Effects of some antibiotics on enzyme activity of glucose-6-phosphate dehydrogenase from human erythrocytes. Pharmacological Research. 41(1):109-113.
8. Çoban Ç, Çiftci M, 2022. Bazı ilaçların koyun dalak dokusundan saflaştırılan glukoz-6-fosfat dehidrogenaz enzimi üzerine in vitro etkileri. Türk Doğa ve Fen Dergisi, 11(1): 29-35.

9. Danner J, Lenhoff HM, Heagy W, 1977. Affinity chromatography of glutathione reductase: Bound by immobilized GSSG, eluted by NADPH. Analytical Biochemistry, (82):586-590.
10. Demir H, Erat M, Şakiroğlu H, 2006. In vitro effects of some antibiotics on glutathione reductase obteined from chicken liver. Turkish Journal of Veterinary and Animal Sciences, (30):513-519.
11. Erat M, Ciftci M, 2006. effect of melatonin on enzyme activities of glutathione reductase from human erythrocytes ın vitro and from rat erythrocytes ın vivo European journal of pharmacology, 537: 59–63.
12. Gill SS, Anjum NA, Hasanuzzaman M, Gill R, Trivedi DK, Ahmad I, Tuteja N, 2013. Glutathione and glutathione reductase: a boon in disguise for plant abiotic stress defense operations. Plant Physiology and Biochemistry, (70):204-212.
13. Knapen MF, Zusterzeel PL, Peters WH, Steegers EA, 1999. Glutathione and glutathione- related enzymes in reproduction, European Journal of Obstetrics & Gynecology and Reproductive Biology, (82):171-184.
14. Karaman, M, Temel Y, Bayindir S, 2020. Inhibition effect of rhodanines containing benzene moieties on pentose phosphate pathway enzymes and molecular docking. Journal of Molecular Structure, 1220, 128700.
15. Keha EE, Küfrevioğlu Öİ. Biyokimya, Aktif Yayınevi, 6. Baskı, İstanbul, 2010.
16. Laemmli UK. 1970 Nature Publishing Group. Nat Publ Gr. 1970;228:1979.
17. Şentürk M. Kufrevioglu Öİ, Çiftci M, 2009. Effects of Some Analgesic Anaesthetic Drugs on Human Erythrocyte Glutathione Reductase: An In Vitro Study. J. Enz. Inh. Med.Chem 24(2): 420–424
18. Taşer P, Ciftci M, 2012. Purification and characterization of glutathione reductase from turkey liver. Turkish Journal of Veterinary and Animal Sciences, 36(5): 546-553.
19. Temel Y, Bozkuş T, Karagözoğlu Y, Çiftçi M, 2017a. Glutatyon redüktaz (GR) enziminin japon bıldırcın (Coturnix coturnix japanica) eritrositlerinden saflaştırılması ve karakterizasyonu. Journal of the Institute of Science and Technology, 7.3: 143-150.
20. Temel Y, Ayna A, Hamdi Shafeeq, I, Ciftci M, 2020. In vitro effects of some antibiotics on glucose-6-phosphate dehydrogenase from rat (Rattus norvegicus) erythrocyte. Drug and chemical toxicology, 43(2), 219-223.
21. Temel Y, Kocyigit U M, 2017. Purification of glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity. Journal of biochemical and molecular toxicology, 31(9), e21927.
22. Temel Y, Küfrevioğlu Öİ, Ciftci M, 2017b. Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity. Turkish Journal of Chemistry, 41(1), 48-60.
23. Temel Y, Taysi MŞ, 2019. The effect of mercury chloride and boric acid on rat erythrocyte enzymes. Biological Trace Element Research, 191(1), 177-182.
24. Temel Y, Bengü AŞ, Akkoyun HT, Akkoyun M, Ciftci M, 2017c. Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats. Journal of Biochemical And Molecular Toxicology, 31(10), e21954.
25. Temel Y, Taher SSM, Hamza MA, Shafeeq İH, Koçyiğit ÜM, Çiftçi M. 2018. Investigation of the inhibition effects of some metal ions on glutathione reductase enzyme from japanese quail (Coturnix coturnix japonica) liver. Cumhuriyet Science Journal, 39(3): 679-687.
26. Ungprasert P, Cheungpasitporn W, Crowson CS, Matteson EL, 2015. Individual Non-Steroidal Anti Inflammatory Drugs and Risk of Acute Kidney Injury: A Systematic Review and Meta-Analysis of Observational Studies. European Journal International Medicine, (26):285-291.
27. Zhang X, Donnan PT, Bell S, Guthriel B, 2017. Non-Steroidal anti-inflammatory drug induced acute kidney injury in the community dwelling general population and people with chronic kidney disease: systematic review and meta-analysis. BMC Nephrology;(18):256.
28. Zhu Y, Zhang QY, 2012. Role of intestinal cytochrome P450 enzymes in diclofenac-induced toxicity in the small intestine. Journal of Pharmacology and Experimental Therapeutics, 343(2):362-370.