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...

Objective-Resistin-like molecule (RELM) β is a secretory protein homologous to resistin and reportedly contributes to local immune response regulation in gut and bronchial epithelial cells. However, we found that activated macrophages also express RELMβ and thus investigated the role of RELMβ in the development of atherosclerosis. Approach and Results-It was demonstrated that foam cells in atherosclerotic lesions of the human coronary artery abundantly express RELMβ. RELMβ knockout ( −/− ) and wild-type mice were mated with apolipoprotein E-deficient background mice. RELMβ −/− apolipoprotein E-deficient mice exhibited less lipid accumulation in the aortic root and wall than RELMβ +/+ apolipoprotein E-deficient mice, without significant changes in serum lipid parameters. In vitro, RELMβ −/− primary cultured peritoneal macrophages (PCPMs) exhibited weaker lipopolysaccharide-induced nuclear factor-κB classical pathway activation and inflammatory cytokine secretion than RELMβ

Resistin-like molecule β (RELMβ) reportedly has multiple functions including local immune responses in the gut. In this study, we investigated the possible contribution of RELMβ to non-alcoholic steatohepatitis (NASH) development. First, RELMβ knock-out (KO) mice were shown to be resistant to methionine-choline deficient (MCD) diet-induced NASH development. Since it was newly revealed that Kupffer cells in the liver express RELMβ and that RELMβ expression levels in the colon and the numbers of RELMβ-positive Kupffer cells were both increased in this model, we carried out further experiments using radiation chimeras between wild-type and RELMβ-KO mice to distinguish between the contributions of RELMβ in these two organs. These experiments revealed the requirement of RELMβ in both organs for full manifestation of NASH, while deletion of each one alone attenuated the development of NASH with reduced serum lipopolysaccharide (LPS) levels. The higher proportion of lactic acid bacteria in the gut microbiota of RELMβ-KO than in that of wild-type mice may be one of the mechanisms underlying the lower serum LPS level the former. These data suggest the contribution of increases in RELMβ in the gut and Kupffer cells to NASH development, raising the possibility of RELMβ being a novel therapeutic target for NASH.