Enzymatic formation of prostaglandin F2 alpha from prostaglandin H2 and D2. Purification and properties of prostaglandin F synthetase from bovine lung.

K Watanabe,R Yoshida,T Shimizu,O Hayaishi

doi:10.1016/s0021-9258(18)88884-6

Abstract

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.

Highlights

Prostaglandin F synthetase from bovine lung was purified 540-fold to apparent homogeneity, as assessed by polyacrylamide gel electrophoreses and ultracentrifugation
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
Phenanthrenequinoneand prostaglandin Dz competitively inhibited the prostaglandin Dz and phenanthrenequinone reductase activities, respectively, these two substrates were albl ut ineffective on the prostaglandin Hz(at the K, value) reductase activity up to 14-fold of those K, values. These results suggest that a single enzyme protein purified from the bovine lung catalyzes the reduction of prostaglandin

Summary

F SYNTHETASEFROMBOVINELUNG*

From the Departmentof Medical Chemistry, Kyoto University Facultyof Medicine, Kyoto606, Japan. Phenanthrenequinoneand prostaglandin Dz competitively inhibited the prostaglandin Dz and phenanthrenequinone reductase activities, respectively, these two substrates were albl ut ineffective on the prostaglandin Hz(at the K , value) reductase activity up to 14-fold of those K , values These results suggest that a single enzyme protein purified from the bovine lung catalyzes the reduction of prostaglandin. We report the purification and characterization of the PGD, 11-ketoreductasefrom bovinelung, the specific activity of which was almost comparable to thatseen in the ratlung This enzyme catalyzes the conversion of PGHz toPGF,, and the reduction of various carbonyl compounds. Evidence is presented that these substrates may be metabolized at two different active sites of a single enzyme This enzyme showed a broad substrate specificity, the best substrate among naturalloyccurring compounds was PGH,. KyotoLaboratory,ResearchDevelopmentCorporation of Japan, on thebasis of partial specific volume of 0.728 ml/g, as Nishioji-Hachijo, Minami-ku, Kyoto601, Japan

Present address

I I I1111

DISCUSSION

EXPERIMENTAL PROCEDURES

Findings

RESULTS