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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 31, Number 2
BKCSDE 31(2)
February 20, 2010 

 
Title
Development of a Coupled Enzyme Assay Method for Microsomal Prostaglandin E Synthase Activity
Author
Kyung A Choi, Sung Jun Park, Yeon Gyu Yu*
Keywords
mPGES-1, 15-PGDH, Fluorescence, NADH, Coupled enzyme assay
Abstract
Human microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes the conversion of prostaglandin H2 (PGH2) into prostaglandin E2 (PGE2). 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, PGH2 was converted to PGE2 by mPGES-1, and then PGE2 was further transformed to the 15-keto-PGE2 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 PGH2 was prevented by PMA. Using this novel assay, the Km value of mPGES-1 for PGH2 and the IC50 value of the previously characterized inhibitor, MK-886, were determined to be 0.150 mM and 2.8 μM, respectively, which were consistent with the previously reported values. In addition, low backgrounds were observed in the multi-wall plate screening of chemical compounds.
Page
384 - 388
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