Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Development of a Coupled Enzyme Assay Method for Microsomal Prostaglandin E Synthase Activity

  • Published : 2010.02.20
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Abstract

Human microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes the conversion of prostaglandin $H_2$ ($PGH_2$) into prostaglandin $E_2$ ($PGE_2$). To establish a stable and efficient method to assess the activity of mPGES-1, a coupled enzyme assay system using mPGES-1, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and phosphomolybdic acid (PMA) was developed. In this assay system, $PGH_2$ was converted to $PGE_2$ by mPGES-1, and then $PGE_2$ was further transformed to the 15-keto-$PGE_2$ by 15-PGDH accompanying the production of NADH, which was easily detected by fluorescence spectrometry in a multi-well plate format. During the reaction, spontaneous oxidation of $PGH_2$ was prevented by PMA. Using this novel assay, the $K_m$ value of mPGES-1 for $PGH_2$ and the $IC_{50}$ value of the previously characterized inhibitor, MK-886, were determined to be 0.150 mM and $2.8\;{\mu}M$, respectively, which were consistent with the previously reported values. In addition, low backgrounds were observed in the multi-wall plate screening of chemical compounds.

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References

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