Glutathione transferase (GST) activity assay of purified MPGES1, expressed in <i>Baculovirus</i> infected <i>SF9 cells</i>.

<p>The enzymatic activity of purified MPGES1 was measured by the conjugation of GSH to the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The spontaneous conjugation of CDNB to GSH was subtracted from each enzymatic reaction and the specific activity values were calculated based on the slope of the initial linear portion of the absorbance curve. The GST activity is expressed as mean ± SD from two independent experiment performed in triplicates.</p

Suggested chemical mechanism of PGH₂ isomerization to PGE₂ by MPGES1 and its active site structure highlighting the amino acids altered.

<p>(A) The thiolate of glutathione (GSH) could be stabilized by Arg126 and attack the C9 oxygen of the PGH₂ endoperoxide forming a sulfenic acid ester. An unidentified proton donor protonates the developing C11 oxyanion. This is followed by proton abstraction at C9 via Asp49. A carbonyl forms and the oxygen sulfur bond is broken forming PGE₂. The leaving GSH thiolate could again be stabilized by Arg126. The unidentified proton donor could then take up the proton from Asp49. (B) The reaction starts by proton abstraction at C9 via Asp49. A carbonyl forms and the endoperoxide bridge is broken. The thiol of GSH functions as a proton donor to the developing C11 oxyanion. After that the proton taken up by Asp49 can reprotonate the GSH thiolate. (C) The interaction between MPGES1 and GSH highlighting the positions of Asp49, Arg126 as well as Ser127 that was proposed to stabilize the GSH thiolate.</p

Prostanoid profiles for WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu, Ser127Ala, expressed in <i>E</i>. <i>Coli</i>.

<p>Prostanoid production was measured by LC-MS/MS after 60 seconds incubations of membrane fractions with PGH₂ (10μM final concentration) and GSH (2.5mM final concentration) at room temperature. Denatured enzymes by boiling were incorporated in the activity assay as controls. All prostanoid measurements of membrane fractions are expressed as mean ± SD from two independent experiment performed in duplicates.</p

Western Blot analysis of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu, Ser127Ala, expressed in <i>E</i>. <i>Coli</i>.

<p>40μg of membrane fraction was loaded into each well. The exposure time was 5 minutes. As a positive control purified MPGES1 was loaded at different concentrations, yielding: 75ng, 100ng, 150ng, 200ng and 450ng.</p

Specific activity of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu and Ser127Ala, expressed and measured in <i>E</i>. <i>Coli</i> membrane fractions.

<p>Specific activity of WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu and Ser127Ala, expressed and measured in <i>E</i>. <i>Coli</i> membrane fractions.</p