3H]prostaglandin F 2α membrane binding reexamined

Abstract

Tritiated prostaglandin F 2α ([ 3H]PGF 2α) binding to bovine corpora luteal membranes has been reexamined from the viewpoint of eventual PGF 2α receptor purification. Several modifications of the literature on PGF 2α binding allow for a more stabilized [ 3H]PGF 2αPGF 2α receptor complex which should then facilitate the PGF 2α receptor purification. Of particular importance were: identification of protease inhibitors which protect [ 3H]PGF 2α binding and protease inhibitors which are detrimental to subsequent [ 3H]PGF 2α binding; the finding that EGTA treatment of tissue homogenates greatly protects subsequent [ 3H]PGF 2α binding; the observation that Mn ++ substitutes for Ca ++ and, in fact, among the divalent cations Mn ++>Mg ++>Ca ++ in facilitating [ 3H]PGF 2α binding where as Cd ++, Cu ++ and Zn ++ either have no effect or are detrimental to this binding; the lack of effect of ATP, GTP, GDP and cAMP or of kinase and phosphatase inhibitors and activators to alter binding of [ 3H]PGF 2α to isolated membranes; and the ease with which the [ 3H]PGF 2α-PGF 2α receptor complex can be removed from the membrane in spite of the receptor being an integral membrane protein. A new simple technique for separating protein bound [ 3H]PGF 2α (PGF 2α receptor-[ 3H]PGF 2α complexes) from free [ 3H]PGF 2α by use of hydroxyapatite (HAP) is introduced. This HAP method is of particular use in solubilized membrane preparations (but can also be used during PG radioimmunoassays to separate free PG from antibody bound PG). These changes were required to facilitate subsequent chromatographic steps leading to identification and purification of the PGF 2α receptor. Finally, a survey of a number of tissue; is performed, using the above mentioned changes and new technique, to access [ 3H]PGF 2α binding. This survey reaffirms that the ovary should be the tissue of choice for isolation of the PGF 2α receptor.