Cloning and heterologous expression of cDNA encoding class alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic alpha,beta-unsaturated carbonyl compounds

Abstract

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha,beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.