engleski

Characterization of Glutathione-S-transferases in zebrafish (Danio rerio)

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish Gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of Gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic GST enzymes after protein purification and overexpression in E. coli. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of Gstp, Gstm (except in liver), Gstr1, Mgst3a and Mgst3b, high expression of Gsto2 in gills and ovaries, Gsta in intestine and testes, Gstt1a in liver, and Gstz in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates, 1- chloro-2, 4- dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity towards CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity towards MCB was in the range of 5 µM (Gstt1a) to 250 µM (Gstp2). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25 s-1 (Gstt1a) to 112 s-1 (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gst pi, Gstt1a, Gstz, Gstr and Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gsta, Gstm, Gst pi, Gstz and Gstr are involved in the crucial physiological processes. In summary, this study provides the first comprehensive analysis of Gst superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts.

Glutathione-S-transferases ; zebrafish ; phylogenetic analysis ; mRNA expression ; enzyme kinetics ; functional characterization

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