The tritium recovery assay of 9-hydroxyprostaglandin dehydrogenase [Pace-Asciak, C. (1975) J. Biol. Chem. 250, 27891 has been modified to ensure its applicability to both crude and purified enzyme preparations. The stereospecificity of NAD+-dependent 9-hydroxyprostaglandin dehydrogenase with respect to NAD+ was determined first and found to be A-side specific. Based on the stereospecificity of the enzyme, a simple and sensitive assay method for 9hydroxyprostaglandin dehydrogenase has been developed. The assay is able to detect picomole quantities of substrate conversion. When 15-keto-13,14-dihydro-[98-3H]PGF,, is employed as substrate, the tritium label of the tritiated prostaglandin is effected to transfer to lactate stereospecifically by coupling 9-hydroxyprostaglandin dehydrogenase with a saturating level of lactate dehydrogenase. The amount of prostaglandin oxidized is quantitated by the radioactivity of the labeled lactate produced, which is separated from labeled prostaglandin by charcoal precipitation. Simultaneous assays with the current tritium-release and thin-layer chromatography methods indicated excellent correlation. Using this method we have found that rat kidney possesses the highest enzyme activity among those tissues examined. Rat kidney enzyme activity is linear for the first 10 min it is studied and is nonlinear with increasing amounts of crude enzyme extract, indicating the possible presence of endogenous inhibitor(s). The apparent K, for 15keto-13,14-dihydro-PGF,, is 0.66 FM. The enzyme is activated by imipramine, inhibited by indomethacin, but not affected by furosemide and ethacrynic acid. These results confirm previous tindings reported in the literature. 9-Hydroxyprostaglandin dehydrogenase catalyzes NAD+-dependent oxidation of the 9a-hydroxyl group of 15-keto-13,14-dihydro-PGF,,' to 15keto-13,lCdihydro-PGE,.
The enzyme was first described by Pace-Asciak (1) who studied the catabolism of PGFzd in rat kidney homogenates and found that the enzyme was responsible for the final conversion to 15-keto-13,1Cdihydro-PGE,.
Studies on the activity . .