CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASES FROM NEEDLES OF Pinus brutia Ten. TREES

Date

2013-01-01

Author

Oztetik, Elif
İşcan, Mesude

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Glutathione S-transferases (GST, E.C. 2.5.1.18) are generally dimeric and multifunctional enzyme family which catalyse the nucleophilic attack of the glutathione on lipophilic compounds with electrophilic centres. Since the 70's GSTs in plant species have been intensively studied, as their role discovered in herbicide detoxification. However, there is only a limited number of studies considering the GST enzyme composition from forest trees, especially not in Pinus brutia, Ten. The trees that exhibited healthy appearance were selected and all belong to the same altitude profile which is located in METU / Yalincak area (Ankara, Turkey). GST activities in the supernatant fractions prepared from needles of P.brutia were determined spectrophotometrically by using 1-chloro-2,4-dinitrobenzene, 2,3-dichloro-4-(2-methylene butyryl)-phenoxy acetic acid (ethacrynic acid), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(p-nitrophenoxy) propane and p-nitrobenzyl chloride as substrates. Only 1-chloro-2,4-dinitrobenzene (160 +/- 10 nmoles min(-1) mg(-1)) and 1,2-dichloro-4-nitrobenzene (2.30 +/- 0.38 nmoles min(-1) mg(-1)) activities were detected and the rest were found as negligible. Accordingly, during purification of GSTs from needles of P.brutia, 1-chloro-2,4-dinitrobenzene was used as the substrate. Purification of GSTs was performed by sequential application of supernatant to gel filtration column chromatography on Sephadex G-25, anion exchange diethylaminoethyl cellulose column chromatography and S-hexylglutathione agarose affinity chromatography. After the final step of purification procedure, 1-chloro-2,4-dinitrobenzene conjugating activity of P. brutia cytosolic GSTs was purified about 15.45 fold with 1.95% yield. Sodium dodecyl sulfate polyacrylamide gel electophoresis results showed that the purified GST isozyme had an Mr of 24 kDa. With this study, we report for the first time the GST isozymes in a gymnosperm, P. brutia.

Subject Keywords

Glutathione S-transferases, Isozymes, Pinus brutia, 1-chloro-2,4-dinitrobenzene, Characterization, Chromatography

URI

https://hdl.handle.net/11511/53962

Journal

FRESENIUS ENVIRONMENTAL BULLETIN

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Department of Biology, Article

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Citation Formats

E. Oztetik and M. İşcan, “CHARACTERIZATION OF GLUTATHIONE S-TRANSFERASES FROM NEEDLES OF Pinus brutia Ten. TREES,” FRESENIUS ENVIRONMENTAL BULLETIN, pp. 516–523, 2013, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/53962.