Fikret TÜRKAN, Neslihan BALCI, Halis ŞAKİROĞLU

KARAYEMİŞ MEYVESİNDEN (Laurocerasus officinalis Roem.) GLUTATYON S-TRANSFERAZ ENZİMİNİN SAFLAŞTIRILMASI VE KARAKTERİZASYONU VE BAZIPESTİSİTLERİN ENZİM AKTİVİTESİNE İNHİBİSYON ETKİLERİ

Bu çalışmada karayemiş (Laurucerasus officinalis Roem.) meyvesinden Glutatyon S- Transferaz (EC 2.5.1.18) enzimi saflaştırıldı ve karakterize edildi. Saflaştırma işlemi tek basamakta afinite kromatografisiyle yapıldı. GST enziminin saflığım kontrol etmek için SDS-PAGE elektroforezi uygulandı. Enzim ile ilgili kinetik çalışmalar olarak optimum pH, optimum sıcaklık, optimum iyonik şiddet ile indirgenmiş glutatyon (GSH) ve 1- kloro 2,4- dinitrobenzen (CDNB) için KM ve V^ çalışmaları yapıldı. Çalışma sonuçlarına göre GSH için KMve V^ değerleri 0.194 mM ve 0.038 EU/ml, CDNB için KMve V^ değerleri 0.353 mM ve 0.099 EU/ml olarak bulundu. Enzim aktivitesi üzerine bazı pestisitlerin etkisi araştırıldı. Bu pestisitlerden; diklorvos, cypermethrin, imidacloprid, fenoxaprop-p-ethyl, glyphosate isopropilamin tuzunun inhibitör özelliği gösterdiği; haloxyfop-p-methyl, lambda- cyhalothrin ve 2,4- dichlorophenoxyasetic asit dimethylamine tuzunun ise aktivatör olarak davrandığı tespit edildi.

Anahtar Kelimeler:

Glutatyon S-Transferaz, Glutatyon, Inhibisyon, Saflaştırma

PDF

___

[1]. Var, M. Seed Fatty Acıd Composıtıon In Wıld Form And Cultıvars Of Laurocerasos Offıcınalıs Roem. Pakistan Journal of Botany, 27(2), 305-308, 1995. [2]. Baytop, T., Türkiye'de Zehirli Bitkiler, Bitki Zehirlenmeleri ve Tedavi Yöntemleri; Yayın 3 560; Istanbul Üniversitesi Yayinlan: Istanbul, Turkey, 1989. [3]. Milan, S.P.A. The Macdonald Encyclopedia of Medicinal Plants; Macdonald: London, U.K. p 252, 1984. [4]. Ayaz, F.A., Studies on Water Soluble Sugar and Sugar Alcohol in Cultivar and Wild Forms of Laurocerasus Offlcinalis Roem, Pakistan J. Bot., 29(2), 331-336, 1997. [5]. Robards, K., Prenzlere, P.D., Tucker, G., Swatsitang, P., Glover, W., Phenolic Compounds and Their Role in Oxidative Processes in Fruits, Food Chem., 66, 401-436, 1999. [6]. Vaya, J., Belinky, P.A., Aviram, M., AntioxidantCconstituents from Licorice Roots: Isolation, Structure Elucidation and Antioxidative Capacity Toward LDL Oxidation, Free Radical Biol. Med., 23 (2), 302-313, 1997. [7]. Vinson, J.A., Su, X., Zubik L., Bose, P., Phenol Antioxidant Ouality in Food: Fruits, J Agric. Food Chem., 49, 5315-5321, 2001. [8]. Smirnoff, N., The Function andMetabolism of Ascorbic Acid in Plants. Ann. Bot. London, 78, 661-669, 1996. [9]. Storz, G., Imlay, J., Oxidative Stress, Curr. Opin. Microbiol. 2, 188-194, 1999. [10]. Ulubelen, A., Topçu, G., Tan, N., Ölçal, S., Johanson, C, Üçer, M., Birman, H., Tamer, S., Biological Activities of a Turkish Medicinal Plant, Prangos Platychlaena, J. Ethnopharmacol., 45(3), 193-197, 1995. [11]. Yan, X., Murphy, B.T, Hammond, G.B., Vinson, J.A., Neto, C.C., Antioxidant Activities and Antitumor Screening of Extracts from Cranberry Fruit (Vaccinium Macrocarpon), J. Agric. Food Chem., 50, 5844-5846, 2002. [12]. Ranson, H., Rossiter, L., Ortelli, F, Jensen, B., Wang, X.L., Roth, C.W, Collins, F.H., Hemingway, J., Identification of a Novel Class of Insect Glutathione S-Transferases Involved in Resistance to ddt in the Malaria Vector Anopheles gambiae, Biochem. 359, 295-304, 2001. [13]. Lumjuan, N., Rajatıleka, S., Changsom, D., Wıcheer, J., Leelapat, P, Prapanthadara, L., Somboon, P, Lycett, G., and Ranson, H., The Role of the Aedes Ring Resistance to DDT and Pyrethroid Insectidies, Insect. Biochem. Mol. Biol, 41, 203-209, 2011. [14]. Furtado, A.F., Magalhaes, T, Macoris, M.L.G., and Righetti, M.T M., Ayres, C.F.J., Resistance to the Organophosphate Temephos: Mechanisms, Evolution and Reversion in an Aedes Aegypti Laboratory Strain from Brazil. Acta. Trop. 113, 180-189, 2010. [15]. Low, W.Y, S.C., Ng, H.L., Gorman, M.A., Morton, C J., Pyke, J., Mecconville, M.J., Bieri, M., Mok, Y.F., Robin, C, Gooley, P R., Parker, M.W., and Batterham, P, Recognotion and Detoxifıcation of the Insecticide DDT by Drosophila Melanogaster Glutathione S-Transferase, J. Mol. Biol, 399, 358-366, 2010. [16]. Clark, A.G., The Glutathione S-trasferases and Resistance to Insecticides, pp. 369-378 InJ. D. Hayes, CB.,PickettandT.J.,Mantle [eds.]. Glutathione S-transferases and Drug Resistance. Proc. 3rd Int. GST Conferance, Royal College of Physicians, Edinburgh, Scotland, UK, 20-30 Aug 1989. [17]. Fukami, J.I., Shishido, T. Nature of a Soluble Glutathione-Dependent Enzyme System Active in Cleavage of Methyl Parathion to Desmethyl Parathion, J. Econ. Entomol, 59, 1338-1346, 1996. [18]. Edwards R, Dixon D.P, Walbot, V, Plant Glutathione S-transferases: Enzymes with Multiple Functions in Sickness and in Health, Trends Plant Sci., 5, 193-198, 2000. [19]. Dixon, D. P., Davis, B. G., Edwards, R. Functional divergence in the glutathione transferase superfamily in plants identification of two classes with putative functions in redox homeostasis in Arabidopsis thaliana. Journal of Biological Chemistry, 277(34), 30859-30869, 2002. [20]. Banerjee, S., Goswami, R. GST profile expression study in some selected plants: in silico approach. Molecular and cellular biochemistry, 336(1-2), 109-126, 2010. [21]. Soranzo, N., Gorla, M. S., Mizzi, L., De Toma, G., Frova, C. Organisation and structural evolution of the rice glutathione S-transferase gene family. Molecular genetics and genomics, 271(5), 511-521, 2004.
[22]. Erat, M., Guvercin, S., Sakiroglu, H. Determination of some kinetic and characteristic properties of glutathione S-transferase from bovine erythrocytes. Protein and peptide letters, 15(1), 6-12, 2008.
[23]. Akkemik, E., Taser, R, Bayindir, A., Budak, H., Ciftci, M., Environmental Toxicology an Pharmacology, 34, 888-894, 2012.
[24]. Chun-hua, Z., Ze-ying, W., Ting, J., Ying, G. Rice Science, 20(3), 173-178, 2013.
[25]. Toribio, F., Martinez-Lara, E., Pascual, P., Lopez-Barea, J. Methods for purification of glutathione peroxidase and related enzymes. Journal of Chromatography B: Biomedical Sciences and Applications, 684(1), 77-97, 1996.
[26]. Simons, P.C., Vander Jagt, D.L. Methods Enzymol, 11 ,235-241, 1981. [27]. Habig, W.H., Pabst, M.J., Jakoby, W.B., Glutathione S-transferases. The first enzymmatic step in mercapturic acid formation. J Biol Chem, 246, 7130-7139, 1974.
[28]. Bradford M.A. Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem., 72, 248-254, 1976.
[29]. Awasthi, Y.C, Singh, S.V., Purification and Characterization of a New form of Glutathione S-Transferase from Human-Erythrocytes. Biochem. Biophys. Res. Commun, 125, 1053-1060, 1984.
[30]. Demirdag, R., Yerlikaya, E., Kufrevioglu, O.I., Gundogdu C, Purification of Glutathione S-transferase Isoenzymes from Tumour and Nontumour Human Stomach and Inhibitory Effects of Some Heavy Metals on Enzymes Activities, Journal of Enzyme Inhibition and Medicinal Chemistry, 28(5), 911-915, 2013.