dinoprost and 9-10-phenanthrenequinone

Prostaglandin (PG)E2 9-ketoreductase, which catalyzes the conversion of PGE2 to PGF2 alpha, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE2, and turnover number were 34,000, 8.2, 6.5-7.5, 1.0 mM, and 7.6 min-1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA2, PGE1, and 13,14-dihydro-15-keto PGF2 alpha, but not that of PGD2, 11 beta-PGE2, PGH2, PGJ2, or delta 12-PGJ2. The reaction product formed from PGE2 was identified as PGF2 alpha by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE2 was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE2 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF2 alpha in human brain.

Topics: Amino Acids; Binding Sites; Brain; Catalysis; Dinoprost; Dinoprostone; Ethacrynic Acid; Humans; Hydrogen-Ion Concentration; Hydroxyprostaglandin Dehydrogenases; Indomethacin; Isoelectric Point; Molecular Weight; NADP; Phenanthrenes; Prostaglandins; Substrate Specificity; Triazines; Vitamin K

Prostaglandin F synthetase from bovine lung was purified 540-fold to apparent homogeneity, as assessed by polyacrylamide gel electrophoreses and ultracentrifugation. The purified enzyme proved to be a monomeric protein with a molecular weight of about 30,500. The enzyme catalyzed not only the reduction of the 11-keto group of prostaglandin D2 but also the reduction of 9,11-endoperoxide of prostaglandin H2 and various carbonyl compounds (e.g. phenanthrenequinone). Experiments using column chromatography, polyacrylamide gel electrophoreses, immunotitration using antibody against the purified enzyme, and heat treatment indicated that three enzyme activities resided in a single protein. Although phenanthrenequinone and prostaglandin D2 competitively inhibited the prostaglandin D2 and phenanthrenequinone reductase activities, respectively, these two substrates were all but ineffective on the prostaglandin H2 (at the Km value) reductase activity up to 14-fold of those Km values. These results suggest that a single enzyme protein purified from the bovine lung catalyzes the reduction of prostaglandin D2, prostaglandin H2, and various carbonyl compounds and that prostaglandin D2 and prostaglandin H2 are metabolized at two different active sites, yielding prostaglandin F2 alpha as the reaction product.

Topics: Amino Acids; Animals; Binding Sites; Cattle; Chromatography, Gel; Dinoprost; Hydroxyprostaglandin Dehydrogenases; Lung; Phenanthrenes; Prostaglandin D2; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins D; Prostaglandins F; Prostaglandins H; Substrate Specificity