Abstract
Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 micromol min-1 mg-1) and high kcat/Km (310 mm-1 s-1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 micromol min-1 mg-1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 micromol min-1 mg-1), 5-hydroperoxyeicosatetraenoic acid (0.043 micromol min-1 mg-1), and 15-hydroperoxy-PGE2 (0.04 micromol min-1 mg-1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 micromol min-1 mg-1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.
Highlights
Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography
MGST1 is closely related to mPGES-1, it is, despite its broad substrate specificity, unable to catalyze the conversion of PGH2 to PGE2, and as yet no functional similarities
Histidine-tagged human mPGES-1 was expressed in E. coli BL21(DE3) identically as described for the non-tagged protein (17)
Summary
Materials—Rabbit polyclonal peptide antiserum directed against mPGES-1 was prepared as described previously (17). Preparation and Solubilization of Whole Cell Extract—A frozen cell pellet from a 1-liter His6-mPGES-1 expression culture was thawed and resuspended in 20 ml of 10 mM sodium phosphate buffer, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM GSH These cells were lysed upon freezethawing by the extrusion of the internal T7 lysozyme as described above. Solubilized membrane fraction/whole cell lysate was mixed with hydroxyapatite (1 g/100 mg of membrane protein or liter expression culture) that had been equilibrated with 10 mM sodium phosphate buffer, pH 8.0, 150 mM NaCl, 1 mM GSH, 10% glycerol, 10 mM imidazole, 0.2% reduced Triton X-100. PGES Activity Assay—PGES activity was assayed in 100 l of reaction mixture, containing 0.1 M potassium phosphate buffer, pH 7.4, 2.5 mM GSH, 10 – 400 M PGH2 (dissolved in acetone), and 0.5 g/ml purified His6-mPGES-1, as described previously (20). Western blots and immunodetection using rabbit polyclonal peptide antiserum directed against mPGES-1 was performed as described previously (20)
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