Development of a Coupled Enzyme Assay Method for Microsomal Prostaglandin E Synthase Activity

作者：Choi Kyung A; Park Sung Jun; Yu Yeon Gyu^*

来源：BULLETIN OF THE KOREAN CHEMICAL SOCIETY, 2010, 31(2): 384-388.

DOI：10.5012/bkcs.2010.31.02.384

• mPGES-1
• 15-PGDH
• Fluorescence
• NADH
• Coupled enzyme assay

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.

• 出版日期2010-2-20