Capoeta trutta Böbrek Dokusundan Saflaştırılan Glutatyon Redüktaz Enzim Aktivitesine Bazı Metallerin in vitro İnhibisyon Etkileri

Yıl 2017, , 385 - 394, 01.12.2017

Muammer Kırıcı , Mahinur Kırıcı , Muhammed Atamanalp

https://doi.org/10.17693/yunusae.v17i31121.337640

Öz

Bu çalışmada, glutatyon redüktaz (EC:
1.8.1.7; GR) iç su balıklarından Capoeta
trutta böbrek dokusundan saflaştırılmıştır. Saflaştırma işlemi, homojenatın
hazırlanması, amonyum sülfat çöktürmesi ve 2’,5’-ADP Sepharose 4B afinite
kromatografisi olmak üzere 3 basamakta gerçekleştirilmiştir. Bu yöntemlerle,
enzim 11.91 EÜ/mg protein spesifik aktivite ve %35.4 verimle 794 kat
saflaştırılmıştır. Saflaştırma sonucu elde edilen saf enzimin saflık kontrolü
sodyum dodesilsülfat poliakrilamid jel elektroforezi (SDS-PAGE) ile yapıldı ve
enzimlerin tek band olduğu görülmüştür. Enzimin molekül kütlesi yaklaşık olarak
55 kDa olarak hesaplanmıştır. Ayrıca, balık böbrek dokusundan saflaştırılan
glutatyon redüktaz enzimi üzerine bazı metallerin (Ag⁺, Co⁺²,
Ni⁺², Cu⁺² ve Zn⁺²) in vitro inhibisyon etkileri incelenmiştir. Metallerin K_i
sabiti ve IC₅₀ değerleri sırasıyla Lineweaver-Burk ve Yüzde (%)
Aktivite-[Metal] grafikleri ile hesaplanmıştır. Bu grafiklerden, Ag⁺,
Co⁺², Ni⁺², Cu⁺² ve Zn⁺² metalleri
için IC₅₀ değerleri sırasıyla 0.00078, 0.622, 0.722, 0.073 ve 0.519
mM, ve K_i sabitleri sırasıyla 0.000394 ± 0.0002, 0.235 ± 0.027,
0.279 ± 0.048, 0.026 ± 0.008 ve 0.382 ± 0.024 mM olarak hesaplanmıştır. Sonuç
olarak, Ag⁺ metalinin diğer metallerden daha fazla C. trutta böbrek GR enzimini inhibe
ettiği tespit edilmiştir.

Anahtar Kelimeler

Glutatyon redüktaz, Capoeta trutta, böbrek, inhibitör, saflaştırma

In vitro Inhibition Effects of Some Metal Ions on Glutathione Reductase Purified from Capoeta trutta Kidney

Öz

Glutathione reductase (EC: 1.8.1.7; GR)
was purified from the kidney of the teleost fish Capoeta trutta. The purification procedure consisted of three
steps: preparation of homogenate, ammonium sulfate fractionation and affinity
chromatography on 2’,5’-ADP Sepharose 4B. The enzyme was purified 794-fold with
a yield of 35.4% and a specific activity of 11.91 U/mg proteins. In order to
control enzyme purity, SDS-PAGE was done and showed a single band for enzyme. A
single band was obtained approximately at 55 kDa. In addition, inhibitory
effects of metal ions (Ag⁺, Co⁺², Ni⁺², Cu⁺²
and Zn⁺²) on fish kidney glutathione reductase were investigated. K_i
constants and IC₅₀ values for metal ions were determined by
Lineweaver–Burk graphs and plotting activity % vs. [I], respectively. IC₅₀
values were 0.00078, 0.622, 0.722, 0.073 and 0.519 mM, and K_i
constants were 0.000394 ± 0.0002, 0.235 ± 0.027, 0.279 ± 0.048, 0.026 ± 0.008
and 0.382 ± 0.024 mM for Ag⁺, Co⁺², Ni⁺², Cu⁺²
and Zn⁺², respectively. From these results, we showed that Ag⁺
is the most potent inhibitor of glutathione reductase enzyme.

Anahtar Kelimeler

Glutathione reductase, Capoeta trutta, kidney, inhibitor, purification

Kaynakça

• Adams, S.M. and Greeley M.S. 2000. Ecotoxicological indicators of water quality: using multi-response indicators to assess the health of aquatic ecosystems. Water, Air and Soil Pollution, 123: 103–115. doi:10.1023/A:1005217622959.
• Akerlund, B., Tynell, E., Bratt, G., Bielenstein, M. and Lidman, C. 1997. N-acetylcysteine treatment and the risk of toxic reactions to trimethoprim-sulphamethoxazole in primary pneumocystis carinii prophylaxis in HIV-infected patients. Journal of Infection, 35: 143–147.
• Akkemik, E., Senturk, M., Ozgeris, F.B., Taser, P. and Ciftci, M. 2011. In vitro effects of some drugs on human erythrocyte glutathione reductase. Turkish Journal of Medical Sciences, 41: 235-241. doi:10.3109/14756366.2011.572879.
• Akkemik, E., Taser, P., Bayindir, A., Budak, H. and Ciftci, M. 2012. Purification and characterization of glutathione S-transferase from turkey liver and inhibition effects of some metal ions on enzyme activity. Environmental Toxicology and Pharmacology, 34: 888-894. doi:10.1016/j.etap.2012.08.010.
• Blahová, J., Plhalová, L., Hostovský, M., Divišová, L., Dobšíková, R., Mikulíková, I., Štĕpánová, S. and Svobodová, Z. 2013. Oxidative stress responses in zebrafish Danio rerio after subchronic exposure to atrazine. Food and Chemical Toxicology, 61: 82–85. doi:10.1016/j.fct.2013.02.041.
• Boggaram, V., Brobjer, T., Larson, K. and Mannervik, B. 1979. Purification of glutathione reductase from porcine erythrocytes by the use of affinity chromatography on 2′,5′-ADP Sepharose 4B and crystallization of the enzyme. Analytical Biochemistry, 98: 335–340.
• Borkovic-Mitic, S., Pavlovic, S., Perendija, B., Despotovic, S., Gavric, J., Gacic, Z. and Saicic, Z. 2013. Influence of some metal concentrations on the activity of antioxidant enzymes and concentrations of vitamin E and SH-groups in the digestive gland and gills of the freshwater bivalve Unio tumidus from the Serbian part of Sava River. Ecological Indicators, 32: 212–221. doi:10.1016/j.ecolind.2013.03.024.
• Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248–254.
• Canpolat, O. 2013. The determination of some heavy metals and minerals in the tissues and organs of the Capoeta umbla fish species in relation to body size, sex, and age. Ekoloji, 22: 64-72.
• Carlberg, I. and Mannervik, B. 1981. Purification and characterization glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2’,5’-ADP Sepharose 4B. Analytical Biochemistry, 116: 531-536.
• Carlberg, I. and Mannervik, B. 1975. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. Journal of Biological Chemistry, 250: 5475–5480.
• Ceyhun, S.B., Senturk, M., Yerlikaya, E., Erdogan, O., Kufrevioglu, O.I. and Ekinci, D. 2011. Purification and characterization of carbonic anhydrase from the teleost fish Dicentrarchus labrax (European seabass) liver and toxicological effects of metals on enzyme activity. Environmental Toxicology and Pharmacology, 32: 69–74. doi:10.1016/j.etap.2011.03.013.
• Coban, T.A., Senturk, M., Ciftci, M. and Kufrevioglu, O.I. 2007. Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study. Protein Peptide Letters, 14: 1027–1030. doi:10.2174/092986607782541060.
• Comakli, V., Akkemik, E., Ciftci, M. and Kufrevioglu, O.I. 2013. Purification and characterization of glucose 6-phosphate dehydrogenase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the effects of some metal ions on enzyme activity. Toxicology and Industrial Health, 1-9.
• Deneke, S.M. and Fanburg, B.L. 1989. Regulation of cellular glutathione. American Journal of Physiology, 257: 163–173.
• Erat, M., Sakiroglu, H. and Ciftci, M. 2003. Effects of some antibiotics on glutathione reductase from bovine erythrocytes. Veterinary Medicine Czech, 48: 305–312.
• Fernandez, B., Campillo, J.A., Martínez-Gómez, C. and Benedicto, J. 2010. Antioxidant responses in gills of mussel (Mytilus galloprovincialis) as biomarkers of environmental stress along the Spanish Mediterranean coast. Aquatic Toxicology, 99: 186–197. doi:10.1016/j.aquatox.2010.04.013.
• Fulladosa, E., Deane, E., Ng, A.H., Woo, N.Y., Murat, J.C. and Villaescusa, I. 2006. Stress proteins induced by exposure to sublethal levels of heavy metals in sea bream (Sparus sarba) blood cells. Toxicology in Vitro, 20: 96-100. doi:10.1016/j.tiv.2005.06.005.
• Gorur, F.K., Keser, R., Akçay, N. and Dizman, S. 2012. Radioactivity and heavy metal concentrations of some commercial fish species consumed in the Black Sea Region of Turkey. Chemosphere, 87: 356–361. doi:10.1016/j.chemosphere.2011.12.022.
• Hu, W., Zhi, L., Zhuo, M.Q., Zhu, Q.L., Zheng, J.L., Chen, Q.L., Gong, Y. and Liu, C.X. 2013. Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro. Fish Physiology and Biochemistry, 39: 637–647. doi:10.1007/s10695-012-9726-x.
• Jenner, P. and Olanow, C.W. 1998. Understanding cell death in Parkinson's disease. Annals of Neurology, 44: 72–84. doi:10.1002/ana.410440712.
• Kaya, E.D., Soyut, H. and Beydemir, S. 2013. Carbonic anhydrase activity from the gilthead sea bream (Sparus aurata) liver: the toxicological effects of heavy metals. Environmental Toxicology and Pharmacology, 36: 514-521. doi:10.1016/j.etap.2013.05.019.
• Kelly, S.A., Havrilla, C.M., Brady, T.D., Abramo, K.H. and Levin, E.D. 1998. Oxidative stress in toxicology: established mammalian and emerging piscine model systems. Environmental Health Perspectives, 106: 375–384. doi:10.1289/ehp.98106375.
• Kirici, M., Kirici, M., Beydemir, S. and Atamanalp, M. 2016. Purification of carbonic anhydrase from Capoeta umbla (Heckel, 1843) gills and toxicological effects of some metals on enzyme activity. Turkish Journal of Fisheries and Aquatic Sciences, 16: 169-175.
• Kondo, T., Dale, O.L. and Beutler, E. 1980. Glutathione transport by inside-out vesicles from human erythrocytes. Proceedings of the National Academy of Sciences of the USA, 77: 6359–6362.
• Kucuksezgin, F., Altay, O., Uluturhan, E. and Kontas, A. 2001. Trace metal and organochlorine residue levels in red mullet (Mullus barbatus) from the eastern Aegean, Turkey. Water Research, 35: 2327–2332. doi:10.1016/S0043-1354(00)00504-2.
• Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680–683.
• Le Trang, N., Bhargava, K.K. and Cerami, A. 1983. Purification of glutathione reductase from gerbil liver in two steps. Analytical Biochemistry, 133: 94-99.
• Lewis, M.A., Scott, G.I., Bearden, D.W., Quarles, R.L., Moore, J., Strozier, E.D., Sivertsen, S.K., Dias, A.R. and Sanders, M. 2002. Fish tissue quality in near-coastal areas of the Gulf of Mexico receiving point source discharges. Science of the Total Environment, 284: 249–261. doi:10.1016/S0048-9697(01)00891-9.
• MacFarlane, G.B. and Burchett, M.D. 2000. Cellular distribution of Cu, Pb, and Zn in the Grey Mangrove Avicemnia marina (Forsk.) Vierh. Aquatic Botany, 68: 45–59. doi:10.1016/S0304-3770(00)00105-4.
• Nisbet, C., Terzi, G., Pilgir, O. and Sarac, N. 2010. Determination of heavy metal levels in fish samples collected from the Middle Black Sea. Journal of the Faculty of Veterinary Medicine, Kafkas University, 16: 119-125.
• Ozaslan, M.S., Demir, Y., Kufrevioglu, O.I. and Ciftci, M. 2017. Some metals inhibit the Glutathione S-transferase from Van Lake fish gills. Journal of Biochemical and Molecular Toxicology, e21967. doi:10.1002/jbt.21967.
• Qu, R., Feng, M., Wang, X., Qin, L., Wang, C., Wang, Z. and Wang, L. 2014. Metal accumulation and oxidative stress biomarkers in liver of freshwater fish Carassius auratus following in vivo exposure to waterborne zinc under different pH values. Aquatic Toxicology, 150: 9–16. doi:10.1016/j.aquatox.2014.02.008.
• Sahin, A., Senturk, M., Akkemik, E. and Ciftci, M. 2012. The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glutathione reductase activity. Nuclear Medicine and Biology, 39: 161–165. doi:10.1016/j.nucmedbio.2011.06.002.
• Sampaio, F.G., Boijink, C.L., Oba, E.T., Santos, L.R.B., Kalinin, A.L. and Rantin, F.T. 2008. Antioxidant defenses and biochemical changes in pacu (Piaractus mesopotamicus) in response to single and combined copper and hypoxia exposure. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 147: 43–51. doi:10.1016/j.cbpc.2012.07.002.
• Schirmer, R.H., Krauth-Siegel, R.L. and Schulz, G.E. 1989. Glutathione reductase. John Wiley and Sons Press, New York.
• Skaggs, H.S. and Henry R.P. 2002. Inhibition of carbonic anhydrase in the gills of two euryhaline crabs, Callinectes sapidus and Carcinus maenas, by heavy metals. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 133: 605–612. doi:10.1016/S1532-0456(02)00175-8.
• Soyut, H. and Beydemir S. 2011. The impact of heavy metals on the activity of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) kidney. Toxicology and Industrial Health, 28: 296-305. doi:10.1177/0748233711410914.
• Soyut, H., Beydemir, S. and Hisar, O. 2008. Effects of some metals on carbonic anhydrase from brains of rainbow trout. Biological Trace Element Research, 123: 179-190. doi:10.1007/s12011-008-8108-9.
• Taser, P. and Ciftci, M. 2012. Purification and characterization of glutathione reductase from turkey liver. Turkish Journal of Veterinary and Animal, 36: 546-553. doi:10.3906/vet-1103-5.
• Tekman, B., Ozdemir, H., Senturk, M. and Ciftci, M. 2008. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 148: 117–121. doi:10.1016/j.cbpc.2008.04.005.
• Turkmen, A., Turkmen, M., Tepe, Y. and Akyurt, I. 2005. Heavy metals in three commercially valuable fish species from İskenderun Bay, Northern East Mediterranean Sea, Turkey. Food Chemistry, 91: 167–172. doi:10.1016/j.foodchem.2004.08.008.
• Ulusu, N.N. and Tandogan, B. 2007. Purification and kinetic properties of glutathione reductase from bovine liver. Molecular and Cellular Biochemistry, 303: 45–51. doi:10.1007/s11010-007-9454-1
• Ulusu, N.N., Tandogan, B. and Tezcan E.F. 2005. Kinetic properties of glucose 6-phosphate dehydrogenase from lamb kidney cortex. Biochimie, 87: 187–190. doi:10.1016/j.biochi.2004.11.002.
• Vijayalingam, S., Parthiban, A., Shanmugasundaram, K.R. and Mohan, V. 1996. Abnormal antioxidant status in impaired glucose tolerance and noninsulin-dependent diabetes mellitus. Diabetic Medicine, 13: 715–719.
• Yonar, M.E. and Sakin, F. 2011. Ameliorative effect of lycopene on antioxidant status in Cyprinus carpio during pyrethroid deltamethrin exposure. Pesticide Biochemistry and Physiology, 99: 226–231. doi:10.1016/j.pestbp.2010.12.008.
• Yonar, S.M., Ural, M.S., Silici, S. and Yonar, M.E. 2014. Malathion-induced changes in the haematological profile, the immune response, and the oxidative/antioxidant status of Cyprinus carpio carpio: Protective role of propolis. Ecotoxicology and Environmental Safety, 102: 202–209. doi:10.1016/j.ecoenv.2014.01.007.
• Zhuo, M., Luo, Z., Tan, X., Hu, W., Sun, L., Zheng, J. and Chan, Q. 2015. Purification and kinetic characteristics of hepatic 6-phosphogluconate dehydrogenase (6PGD) from yellow catfish Pelteobagrus fulvidraco. Turkish Journal of Biochemistry, 40: 15–23. doi:10.5505/tjb.2015.75537.