[13] Purification of PGH-PGE isomerase from sheep vesicular glands

Publisher Summary This chapter presents a procedure for the purification of prostaglandin (PG) H–PGE isomerase from sheep vesicular glands. In the purification procedure described in the chapter, deep-frozen sheep seminal vesicles, trimmed free from fat and connective tissue, were homogenized in 75-g portions with 110 ml of 0.05 M Tris-HCl, pH 8.0, containing 10 mM ethylenediaminetetraacetic acid disodium salt and 1 mM diethyldithiocarbamate using a Sorvall Omnimixer and a VirTis 45 homogenizer. Further, the homogenate was tween-treated. Steps of purification include chromatography on diethylaminoethyl-cellulose and chromatography on hydroxyapatite-agarose. The endoperoxide used as substrate in the assay was not stable in aqueous medium: it has a half-life of about 10 min at pH 7.4 and 20 °. The main degradation products were PGE and PGD in a ratio of about 3:1. Therefore, incubations were carried out for only one min with different amounts of enzyme and with a fixed amount of 14 C-labeled endoperoxide. After quick acidification and extraction with ether, the products formed and the remaining endoperoxide were rapidly separated by silica gel- high performance liquid chromatography. Quantification of the enzyme activity was possible by scraping off the radioactive bands, counting the radioactivity, and calculating the nanomoles of PGE formed.