Abstract
Publisher Summary This chapter presents a procedure for the purification of nicotinamide adenine dinucleotide phosphate (NADP) + /NADPH-dependent 15-hydroxyprostaglandin dehydrogenase and prostaglandin 9-ketoreductase from porcine kidney. In the purification procedure, all steps of purification were performed at 4°. Porcine kidney was homogenized in 1 liter of 10 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA (buffer A) in a Waring blender for 2 min. The homogenate was centrifuged at 40,000 g for 2 min. Further steps were (1) acetone fractionation, (2) sephadex G-100 chromatography, (3) TEAE-cellulose chromatography, and (4) isoelectric focusing. The active fractions were concentrated by lyophilization and dialyzed against buffer for 24 hr. It is suggested that the assay of 15-hydroxyprostaglandin dehydrogenase activity can be done by a spectrophotometric method based on the conversion of PGE to 15-keto-PGE that forms a chromophore after alkalinization of the reaction mixture. The activity can also be assayed spectrofluorometrically by following the increase in fluorescence of NADPH at 460 nm with excitation at 340 nm.
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