Purification and some properties of glutathione S-transferase from Escherichia coli B

Abstract

Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B with a 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist of two homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (K,. 1.43 mM) and glutathione (K,,, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50°C, respectively, and the enzyme was stable from pH 5 to 11. The activity of the enzyme for 1-chloro-2,4-dinitrobenzene (3.2 ,umol/min per mg of protein) was significantly lower than those of the enzymes from mammals, plants, and fungi.Glutathione S-transferases (GSTs) constitute an important class of detoxifying enzymes. The enzyme catalyzes the conjugation of glutathione (GSH) with various compounds having electrophilic and/or hydrophobic sites (6). The enzyme is thought to protect the cells against foreign compounds such as pesticides, drugs, and carcinogens (7). The enzymes have been extensively purified from mammals such as human (8), mouse (12), cattle (3), and rat (21), and their properties have been characterized in detail. However, the data on microbial GSTs are largely lacking, and the enzyme has been purified only from Fusarium oxysporum f. sp. melonis (5), Mucorjavanicus (1), and Tetrahymena thermophila (18). Although attempts to detect GST activity in cell extracts of bacteria (2, 11, 21), yeasts (4, 9), mold (1, 5), and protozoa (18) have been made, these results are fragmentary and not enough to compare the properties of microbial enzymes with those of enzymes from mammals. Here we report the purification and partial characterization of GST from Escherichia coli B.MATERIALS AND METHODS Chemicals. GSH was purchased from Kohjin Co., Ltd., Tokyo, Japan. 1-Chloro-2,4-dinitrobenzene was from Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan. Glutathione agarose was prepared from AH-Sepharose CL-4B according to the vendor's specifications (Pharmacia).Assays. The activity of GST was assayed essentially by the method of Habig et al. (6). The reaction mixture (1.0 ml) consisted of 0.1 M potassium phosphate buffer (KPB) (pH 7.0), 1.0 mM EDTA, 1.0 mM GSH, 1.0 mM 1-chloro-2,4-dinitrobenzene (CDNB), and enzyme. The enzyme activity was calculated by using a molar extinction coefficient of S-(2,4-dinitrophenyl)glutathione as 9.6 M-1 cm-' at 340 nm and 25°C. One unit of enzyme activity was defined as the amount producing 1 nmol of conjugate of GSH with CDNB per min. Protein was determined by the method of Lowry et al. (13). GSH in cells was determined by the method of Murata et al. (17).Growth experiments. In order to investigate the effect of electrophiles on the formation of GST activity, cells of E. coli B were grown in a test tube containing 5 ml of nutrient medium (1.0% glucose, 0.1% yeast extract, 1.0% peptone, 0.5% meat extract, 0.1% MgSO4-7H20, and 0.5% K2HPO4 * Corresponding author.[pH 7.0]) with reciprocal shaking at 37°C for 20 h. The cells were transferred into a 2-liter Sakaguchi flask containing...

Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane

“…In the past, GST had been characterized in eukaryotes as a detoxifying enzyme. However, several recent reports concerning bacterial GSTs have appeared (1,13,24). It has been shown that GST plays a role in the assimilation of dichloromethane in Methylobacterium spp.…”

Bacterial glutathione S-transferases: what are they good for?

“…They are not quantitatively prominent proteins in the cell, judging from the purification factors reported (0.002 to 0.1%) (32,64,71). The presence of CDNB-active GST enzymes was associated with increased resistance of the bacterial host to several antibiotics (70,71).…”

Purification and Biochemical Properties of Glutathione S-Transferase from Lactuca sativa

“…The activity of the enzyme toward CDNB was significantly lower than the enzymes from mouse, corn and F. oxysporum (Lee et al, 1981;Mozer et al, 1983;Ando et al, 1988). On the other hand, it was similar to those of the enzymes from bacteria (Izuka et al, 1989;Nishida et al, 1994).…”

“…The theta-class GSTs purified from Arabidopsis thaliana and mouse liver exhibited selenium-independent glutathione peroxidase activity (Bartling et al, 1993;Hiratsuka et al, 1995). On the other hand, E. coli B GST showed neither selenium-dependent nor independent glutathione peroxidase activity, indicating that the properties of catalytic sites between eukaryote and prokaryote enzymes may be different (Izuka et al, 1989). The molecular cloning of the GST gene of Lactuca sativa is now in progress in order to elucidate the difference in the molecular structure between the Lactuca sativa GST and enzymes of other sources.…”

“…Plant GST has been concerned in the agricultural chemistry and biochemistry because it is one of the major factors involved in the resistance of a variety of herbicides and insecticides (Lamoureux et al, 1980;Leurs et al, 1989). The first GST reported to participate in herbicide metabolism was isolated from maize and characterized in some detail (Mozer et al, 1983;Moore et al, 1986;Grove et al, 1988;Izuka et al, 1989;Irzyk et al, 1993Irzyk et al, , 1995. GSTs play roles in plants, having been implicated in herbicide resistance, being inducible by pathogens and/or dehydration, showing direct binding of auxins and catalyzing the formation of anthocyanins (Sheehan et al, 2001).…”