Continuous spectrophotometric assay amenable to 96-well plate format for prostaglandin E synthase activity.
The measurement of prostaglandin E synthase (PGES) activity is cumbersome because the product of the reaction, PGE(2), is not readily quantitated by spectral means. The activity of isolated PGES is typically determined by PGE(2) immunoassay or by high-performance liquid chromatography using radiolabeled substrate. A relatively rapid continuous spectrophotometric assay which uses 15-hydroxyprostaglandin dehydrogenase (PGDH) to couple the oxidation of the 15-hydroxy group of PGE(2) to the formation of NADH was developed. PGDH is relatively specific for PGE(2) over the substrate for the PGES reaction, PGH(2), allowing a highly reproducible assay of PGES activity to be obtained.
David Percival
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Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Kirkland, Que., Canada H9R 4P8. dave_percival@merck.com
D015232:Dinoprostone / Q000378:metabolism; D006911:Hydroxyprostaglandin Dehydrogenases / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000032:analysis / Q000096:biosynthesis; D044262:Prostaglandin H2; D000071876:Prostaglandin-E Synthases; D011463:Prostaglandins H / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*
D011463:Prostaglandins H; D011993:Recombinant Fusion Proteins; D009243:NAD; D044262:Prostaglandin H2; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D015232:Dinoprostone