[3H]prostaglandin F2 alpha membrane binding reexamined.
Tritiated prostaglandin F2 alpha ([3H]PGF2 alpha) binding to bovine corpora luteal membranes has been reexamined from the viewpoint of eventual PGF2 alpha receptor purification. Several modifications of the literature on PGF2 alpha binding allow for a more stabilized [3H]PGF2 alpha PGF2 alpha receptor complex which should then facilitate the PGF2 alpha receptor purification. Of particular importance were: identification of protease inhibitors which protect [3H]PGF2 alpha binding and protease inhibitors which are detrimental to subsequent [3H]PGF2 alpha binding; the finding that EGTA treatment of tissue homogenates greatly protects subsequent [3H]PGF2 alpha binding; the observation that Mn(+)+ substitutes for Ca(+)+ and, in fact, among the divalent cations Mn(+)+ greater than Mg(+)+ greater than Ca(+)+ in facilitating [3H]PGF2 alpha 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]PGF2 alpha to isolated membranes; and the ease with which the [3H]PGF2 alpha-PGF2 alpha 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]PGF2 alpha (PGF2 alpha receptor-[3H]PGF2 alpha complexes) from free [3H]PGF2 alpha 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 PGF2 alpha receptor. (ABSTRACT TRUNCATED AT 250 WORDS)
Orlicky
D J
DJ
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003338:Corpus Luteum / Q000187:drug effects / Q000378:metabolism*; D015237:Dinoprost / Q000378:metabolism*; D005260:Female; D006868:Hydrolysis; D009711:Nucleotides / Q000494:pharmacology; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D011982:Receptors, Prostaglandin / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium
D002413:Cations, Divalent; D009711:Nucleotides; D011480:Protease Inhibitors; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D014316:Tritium; D015237:Dinoprost