Purification and characterization of recombinant microsomal prostaglandin E synthase-1.
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.
Ouellet
Marc
M
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Kirkland, Que., Canada H9R 4P8.
Falgueyret
Jean-Pierre
JP
Ear
Po Hien
PH
Pen
Ally
A
Mancini
Joseph A
JA
Riendeau
Denis
D
Percival
M David
MD
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
D003902:Detergents; D011994:Recombinant Proteins; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases