Purification of glutathione S-transferases and genetic characterization of Zeta isozyme from Pinus brutia, Ten

Öztetik, Elif
Glutathione S-transferases (GST, EC2.5.1.18) are a family of multifunctional, dimeric enzymes that catalyse the nucleophilic attack of the tripeptide glutathione (?-L-glutamyl-L-cysteinyl-L-glycine) on lipophilic compounds with electrophilic centres. The primary function of GSTs is generally considered to be the detoxification of both endogenous and xenobiotic compounds. Cytosolic GSTs have been grouped into eleven distinct classes as: (A); Alpha, (M); Mu, (P); Pi, (S); Sigma, (T); Theta, (Z); Zeta, (F); Phi, (U); Tau, (B); Beta, (O); Omega and (L); Lambda. iv In this study, the total RNAs from Pinus brutia needles were isolated, GST Zeta cDNA was prepared by RT-PCR, the length of the insert was elongated by applying 5' RACE (Rapid Amplification for cDNA ends) method and the identity of the insert was checked by sequencing. The amino acid sequence of GST-Zeta was deduced as composed of 226 amino acids. The genomic DNA was also isolated from Pinus brutia needles, amplified by PCR and sequenced, and compared to the sequence of cDNA. The expression level of GST-Zeta in individual trees of Pinus brutia were examined by Northern blot analysis, and compared to their thiol contents. The mRNA levels varied up to three-fold, whereas GSH amounts varied approximately 1.8 fold, and there were no correlation between the GST-Zeta expression and GSH concentration. GST enzyme with activity towards CDNB was isolated and purified from Pinus brutia needles in 1.95 % yield with a purification factor of 15.45-fold. The purification protocol included a sequential chromatography on Sephadex G-25 column, DEAE cellulose anion exchanger liquid chromatography column, and S-hexylglutathione agarose affinity columns. The purified GST showed specific activity towards CDNB as 2022 nmole/min/mg. The GST purified from needles had a molecular weight (Mr) value of about 24.000 which was confirmed by


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Citation Formats
E. Öztetik, “Purification of glutathione S-transferases and genetic characterization of Zeta isozyme from Pinus brutia, Ten,” Ph.D. - Doctoral Program, Middle East Technical University, 2005.