Pinus brutia, Ten'den glutatyon S-transferazların saflaştırılması ve zeta izoziminin genetik karakterizasyonu.

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 byPh.D. - Doctoral Progra

Molecular characterization of zeta class glutathione S-transferases from Pinus brutia Ten.

WOS: 000363980200006PubMed: 26440080Glutathione transferases (GSTs; EC 2.5.1.18) play important roles in stress tolerance and metabolic detoxification in plants. In higher plants, studies on GSTs have focussed largely on agricultural plants. There is restricted information about molecular characterization of GSTs in gymnosperms. To date, only tau class GST enzymes have been characterized from some pinus species. For the first time, the present study reports cloning and molecular characterization of two zeta class GST genes, namely PbGSTZ1 and PbGSTZ2 from Pinus brutia Ten., which is an economically important pine native to the eastern Mediterranean region and have to cope with several environmental stress conditions. The PbGSTZ1 gene was isolated from cDNA, whereas PbGSTZ2 was isolated from genomic DNA. Sequence analysis of PbGSTZ1 and PbGSTZ2 revealed the presence of an open reading frame of 226 amino acids with typical consensus sequences of the zeta class plant GSTs. Protein and secondary structure prediction analysis of two zeta class PbGSTZs have shared common features of other plant zeta class GSTs. Genomic clone, PbGSTZ2 gene, is unexpectedly intronless. Extensive sequence analysis of PbGSTZ2, with cDNA clone, PbGSTZ1, revealed 87% identity at nucleotide and 81% identity at amino acid levels with 41 amino acids differences suggesting that genomic PbGSTZ2 gene might be an allelic or a paralogue version of PbGSTZ1.Tubitak; University of Anadolu Reseach ProjectThanks to Prof. Heinz Rennenberg and Dr Stanislav Kopriva from Forest Botanic and Tree Physiology Institute, Albert-Ludwigs University, Freiburg, Germany, for providing the initial EST sequences and their help in Northern blot and glutathione assays. Dr Elif Oztetik was supported by Tubitak short term fellowship. This work was financially supported by University of Anadolu Reseach Project

Molecular cloning and biochemical characterization of a Tau class glutathione S-transferase from Pinus brutia Ten

Alper, Meltem ( Aksaray, Yazar )Key message: A new Tau class GST gene was cloned from Pinus brutia Ten. cDNA sequence was analysed for conserved sequences. Substrate specificity, optimum pH, and temperature values of the recombinant PbGST Tau enzyme were determined. Abstract: Tau class glutathione S-transferases (GSTs) are essential enzymes for detoxification in plants. To date, a lot of the members of this family have been characterized from different plants but the studies on the conifers are very scarce. This study investigates for the first time molecular cloning and biochemical characterization of a Tau class GST gene (PbGST Tau) from Pinus brutia Ten. The full length PbGST Tau ORF was 687 bp having a molecular mass of 27.37 kDa. Catalytic and ligand binding sites of PbGST Tau are well conserved and shared maximum identity with Pinus tabulaeformis GST Tau. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ECA) as substrates exhibited a Km of 3.66 mM and 0.3 mM, respectively. PbGST Tau enzyme had an optimum activity at pH 6.0 and 8.0 when CDNB and ECA were used as substrate, respectively. The highest activity was measured at 25 °C. Through enzyme assays, phylogenetic analysis and structural modelling, we provide a detailed characterization of the PbGST Tau gene and the enzyme. This study is going to provide new insights into the phylogenetic and biochemical analysis of GST family in conifers