Journal of Lipid Research, Vol 26, 713-720, Copyright © 1985 by Lipid Research, Inc.
Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver
PD Roach and SP Noel
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.
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