title: Purification and characterization of recombinant microsomal prostaglandin E synthase-1.
abstract: Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K(m) values of the substrates PGH(2) and GSH were 14 microM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 micromol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V(max) but does not affect the K(m) values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE(2) formation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Kirkland, Que., Canada H9R 4P8.
authors.forename: Marc
authors.initials: M
authors.lastname: Ouellet
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Falgueyret
authors.forename: Po Hien
authors.initials: PH
authors.lastname: Ear
authors.forename: Ally
authors.initials: A
authors.lastname: Pen
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Mancini
authors.forename: Denis
authors.initials: D
authors.lastname: Riendeau
authors.forename: M David
authors.initials: MD
authors.lastname: Percival
mesh_terms: D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D000071876:Prostaglandin-E Synthases; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D018411:Spodoptera
chemical_list: D003902:Detergents; D011994:Recombinant Proteins; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases