title: Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver.
abstract: Pharmacological doses of 17 alpha-ethinyl estradiol induce a low density lipoprotein (LDL) receptor in the liver of male rats. Our aim was to solubilize this receptor. Isolated liver membranes (8,000-100,000 g fraction) from male rats treated with 17 alpha-ethinyl estradiol and from control rats were solubilized in 1% (w/v) Triton X-100. Using Amberlite XAD-2, more than 90% of the detergent was then removed. Liposomes were prepared by precipitating the solubilized proteins with acetone in the presence of phosphatidylcholine. The receptor activity of these liposomes was assayed using human 125I-labeled LDL. Filtration was used to separate bound from free 125I-labeled LDL. The assay was optimized; 0.25 mM CaCl2, 25 mM NaCl, pH 8.0, were chosen as the standard conditions. Binding of 125I-labeled LDL was dependent on Ca2+. Liposomes containing solubilized membrane proteins from treated rats displayed Ca2+-dependent binding which was 11 times higher than for control rats. The specific binding of 125I-labeled LDL was saturable with a Kd = 18 micrograms/ml. 125I-Labeled LDL was displaced by unlabeled lipoproteins containing apolipoproteins B and E and by dimyristoylphosphatidylcholine liposomes containing purified apolipoprotein E, but not by HDL3. The binding was abolished by pronase and was inhibited by suramin. Ligand blotting with 125I-labeled LDL revealed one band of protein with an apparent molecular weight of 133,000 daltons. These properties are characteristic of the low density lipoprotein receptor.
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D004997:Ethinyl Estradiol / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008081:Liposomes; D008099:Liver / Q000032:analysis* / Q000187:drug effects; D008297:Male; D008970:Molecular Weight; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011973:Receptors, LDL / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013498:Suramin / Q000494:pharmacology
chemical_list: D008077:Lipoproteins, LDL; D008081:Liposomes; D011973:Receptors, LDL; D004997:Ethinyl Estradiol; D013498:Suramin