COVALENT BINDING OF PROSTAGLANDIN-G2 AND PROSTAGLANDIN-H2 TO COMPONENTS OF RAM SEMINAL-VESICLE MICROSOMAL FRACTION

Following incubation of 1-14C arachidonic acid with a microsomal preparation of ram seminal vesicles, 90 per cent of the products formed were classical prostaglandins and hydroxyfatty acid. In addition, approximately 8 per cent of the products formed possessed radioactivity which was covalently bound to the microsomal pellet after acid precipitation and solvent extraction. The amount of covalently bound material derived from arachidonate was related to the concentration of free prostaglandin (PG) endoperoxides in the incubation mixture, indicating that these reactive intermediates were probably responsible for the generation of the covalently bound material. Protein-associated radioactivity was also observed following incubation of | 1-14C |PGG2 or | 1-14C |PGH2 with a heat-denatured preparation of ram seminal vesicle microsomes. Such preparations do not convert PGG2 to PGH2; thus, the binding of PGG2 was not due to its prior conversion to PGH2. The rate constant for the covalent binding of PGG2 (0.19 min-1) was approximately twice that for PGH2 (0.11 min-1), suggesting that both 9, 11-endoperoxy and 15-hydroperoxy groups participate in the binding reaction. Moreover, the rate constant of binding derived from arachidonate (0.12 min-1) approximated those of the purified endoperoxides. In an active microsomal preparation, glutathione reduced covalent binding and increased PGE2 formation without greatly affecting oxygen consumption and, therefore, without affecting total product formation from arachidonic acid. Glutathione, therefore, probably inhibited covalent binding by lowering free endoperoxide concentrations in the incubation mixture. Diethyldithiocarbamate and phenylbutazone, but not para-aminophenol or butylated hydroxyanisole, inhibited the covalent binding of PGG2 to heat-denatured tissue protein. These results suggest that the covalent binding reaction may proceed via the formation of free radicals. © 1979.