Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Abstract

Recombinant human microsomal prostaglandin E2 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 Km values of the substrates PGH2 and GSH were 14 μM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 μmol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases Vmax but does not affect the Km 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 PGE2 formation. © 2002 Elsevier Science (USA). All rights reserved.