Enzymatic synthesis of (15s)-[15-3h]prostaglandins and their use in the development of a simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase.
The stereospecificity of swine renal NAD+-dependent 15-hydroxyprostaglandin dehydrogenase has been determined. It was found that the enzyme is a B-side specific dehydrogenase. (15S)-[15-3H]Prostaglandins were synthesized by stereospecific transfer of the tritium label of D-[1-3H]galactose to prostaglandins by coupling 15-hydroxyprostaglandin dehydrogenase with beta-D-galactose dehydrogenase, an enzyme of the same stereospecificity. A simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase was developed based on the stereospecific transfer of the tritium label of tritiated prostaglandins to glutamate by coupling 15-hydroxyprostaglandin dehydrogenase with glutamate dehydrogenase. The amount of prostaglandin oxidized is determined by the radioactivity of labeled glutamate present in the supernatant after charcoal precipitation of labeled prostaglandin. Concurrent assays with the present tritium release method and the thin-layer chromatography method indicated excellent correlation. The assay was employed to study some of the properties of swine renal 15-hydroxyprostaglandin dehydrogenase in crude extract and the distribution of enzyme activity in various tissues of rat. Enzyme activity was linear for the first 10 min studied and was nonlinear with increasing amounts of crude enzyme, indicating the possible presence of endogenous inhibitor(s). Apparent Km's for PGE2, PGF2alpha, and PGA2 were found to be 2.5, 12.5, and 3.9 muM, respectively. The distribution pattern indicated high levels of enzyme activity in gastrointestinal tract, lung, kidney, and spleen. The assay method may prove to be valuable for studying enzyme turnover and enzyme regulation by hormonal and pharmacological agents.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D005692:Galactose Oxidase / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006911:Hydroxyprostaglandin Dehydrogenases / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008722:Methods; D009243:NAD; D009928:Organ Specificity; D011453:Prostaglandins / Q000096:biosynthesis*; D051381:Rats; D013552:Swine
D011453:Prostaglandins; D009243:NAD; D000429:Alcohol Oxidoreductases; D006911:Hydroxyprostaglandin Dehydrogenases; D005692:Galactose Oxidase; D005969:Glutamate Dehydrogenase