25] Isolation and properties of an NADP+-dependent PGI2-specific 15-hydroxyprostaglandin dehydrogenase from rabbit kidney

Abstract

Publisher Summary This chapter presents a procedure for the isolation and assay of nicotinamide adenine dinucleotide phosphate (NADP + )-dependent prostaglandin (PG) I 2 -specific 15-hydroxyprostaglandin dehydrogenase from rabbit kidney. For purification described in the chapter, frozen rabbit kidneys were thawed and minced. Portions of 50–60 g were homogenized with four volumes of buffer in a Waring blender. A 10-ml portion of the homogenate was centrifuged at 30,000 g for 60 min, and the supernatant solution was assayed for enzyme activity. The remainder of the homogenate was centrifuged at 9000 g for 75 min, and the supernatant solution was collected. Further steps were (1) ammonium sulfate fractionation, (2) diethylaminoethyl-cellulose chromatography, (3) matrex gel blue a chromatography, (4) sephadex G-100 gel filtration, and (4) hydroxyapatite chromatography. The enzyme activity was measured spectrophotometrically in a cuvette with a l-cm light path at 25 ±0.5°. The cuvette contains 3 ml of aqueous solution consisting of 290 μmol of sodium pyrophosphate at pH 10.3, 1.25 μmol of NADP + , and 267 nmol of PGI 2 added in 0.01 ml of sodium pyrophosphate. The blank contains no PGI 2 but was identical otherwise. The reaction was started by the addition of enzyme, and NADPH production was followed by measuring the change in absorbance at 340 nm.