A radioimmunoassay for prostaglandin endoperoxide synthetase

A radioimmunoassay for prostaglandin endoperoxide synthetase has been developed. The antiserum was produced in rabbits by repeated immunization with the purified sheep seminal vesicular enzyme. The enzyme was labeled by chloramine T mediated radioiodination and purified by Sephadex G-25 gel filtration followed by hydroxyapatite column chromatography. Separation of bound from free enzyme was achieved by double antibody method. The radioimmunoassay was capable of detecting 1 ng per assay tube. The antiserum was found to crossreact with the enzyme from various animal tissues. Subcellular localization of the enzyme in sheep seminal vesicular gland indicated that distribution of immunoreactive enzyme in subcellular fractions correlated well with that of enzyme activity with highest enrichment in the microsomal fraction. Application of radioimmunoassay to the measurement of immunoreactive enzyme in kidneys of normotensive and genetically hypertensive rats revealed that the level was significantly increased in hypertensive rats at 18 weeks but not at 10 weeks of age. The level of immunoreactive enzyme also correlated well with that of enzyme activity. The availability of a radioimmunoassay will provide a valuable tool for probing the structure and function of the enzyme in various physiological and pathophysiological states.