Prostaglandin Synthetase System—Resolution into Oxygenase and Isomerase Components

Abstract

The microsomal fraction of bovine vesicular gland catalyzed the conversion of [1-¹⁴C]8,11,14-eicosatrienoic acid to prostaglandin E₁ in the presence of tryptophan, hemoglobin, and glutathione. The prostaglandin synthetase system was solubilized by treatment of the microsomal fraction with Tween 20 in the presence of ethylene glycol. DEAE-cellulose column chromatography separated the enzyme into two fractions (Fractions I and II), both of which were required for prostaglandin E₁ synthesis. When Fraction I alone was incubated with 8,11,14-eicosatrienoic acid, an unstable compound accumulated. This compound was converted to prostaglandin E₁ by the addition of Fraction II. On the basis of its R_F values on thin-layer chromatography, its reduction to prostaglandin F_1α with stannous chloride, and the decomposition to prostaglandins F_1α, E₁, and D₁ at room temperature, the unstable intermediate was tentatively identified to be the 9,11-endoperoxide derivative, referred to previously as prostaglandin R₁ (Nugteren et al. (1973) Biochim. Biophys. Acta 326, 448-461) or prostaglandin H₁ (Hamberg et al. (1974) Proc. Nat. Acad. Sci. USA 71, 345-349). Of the three cofactors mentioned above, Fraction I required both tryptophan and hemoglobin, while only glutathione was necessary for Fraction II. Anti-inflammatory agents such as indomethacin and aspirin were inhibitory to Fraction I.