Binding of 17-alpha-ethinyl estradiol and mestranol to uterine and pituitary gland receptors of the rat in vitro

Comparative study, through radiochemical analysis, of the binding of estrogens to the uterine and pituitary receptors in immature rats showed that, for binding to the uterine recpetors, a free OH group in Position 3 is necessary. 17alpha-ethinyl estradiol as well as mestranol (17alpha-ethinyl estradiol-3-methyl ether), showed a stronger affinity for the pituitary than the uterine receptors. Indeed, mestranol, among the 3-methyl ether estrogens, has a special place in respect to its affinity for the pituitary. Since the binding to the receptors is an essential stage in inhibition or stimulation effects, this is a significant point. It may be that mestranol exercises its recognized central effect not only indirectly through release of ethinyl estradiol, but also directly.     

Metabolism of radioactive 17alpha-ethynylestradiol by women

Data on the urinary excretion and subsequent fractionation of radioactivity derived from 6,7-tritiated-17alpha-ethinyl estradiol (tritiated EE) are presented. Tritiated EE was administered orally to 9 women. 17alpha-ethinyl estradiol 2-methoxy-17alpha-ethinyl estradiol, 2 -hydroxy-17alpha-ethinyl estradiol 3-methyl ether, and D-homoestradiol-1 7abeta were identified as urinary metabolites by reverse isotope dilution. The extent of D-homoannulation was much less than that reported previously with rabbits.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.     

Reaction of thiol nucleophiles with 1,2-epoxy- and 4,5-epoxy-estrene-3-one-17 beta-ols

Four ring A steroidal epoxyenones as probable intermediate in the formation of catechol estrogens were synthesized. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one (9) and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (8) were synthesized from 17 beta-hydroxy-5 alpha-estra-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one (11) and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one (10) were prepared from 19-nortestosterone. The reaction of 9 and 10 with sodium/ethanethiol resulted in the formation of three types of reactions leading to multiple products: 1,4-addition, opening of epoxide, and epoxide opening followed by dehydration. Reaction of 8 with ethanethiol gave only one compound identified as 2-ethanethio-1,4-estradien-17 beta-ol-3-one, while reaction of 9 with ethanethiol gave an unusual product identified as 4-estren-1 alpha,17 beta-diol-3-one. Unlike reaction of ethanethiol with 9 and 10, reaction with N-acetylecysteine or glutathione results in epoxide opening followed by dehydration leading to the formation of estradiol-4-thioethers.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Regulation of hepatic apolipoprotein synthesis in the 17 alpha-ethinyl estradiol-treated rat

Regulatory mechanisms of hepatic apolipoprotein synthesis were studied in groups of male Sprague-Dawley rats made severely hypolipidemic by treatment with pharmacological doses of 17 alpha-ethinyl estradiol. Treatment resulted in a marked reduction of plasma cholesterol and apolipoproteins B, A-I, and A-IV. Hepatic apoA-I mRNA and apoA-I synthesis were increased in the ethinyl estradiol-treated animals. Hepatic apoA-IV protein synthesis rates were unaltered; however, a reduction of the apoA-IV mRNA level was observed. Diet-control studies suggested the effects of 17 alpha-ethinyl estradiol on apoA-I, unlike those on apoA-IV, appeared to be related to the steroid and not to reduced caloric intake. Livers of control and ethinyl estradiol-treated rats synthesized both apoBH and apoBL. Total hepatic apoB (apoBL plus apoBH) synthesis and apoB mRNA levels in the ethinyl estradiol-treated rats were similar to ad libitum fed or diet-controls. In ad libitum fed and diet-control rats, 21% and 32%, respectively, of newly synthesized hepatic apoB was apoBH. In contrast, 47% of the newly synthesized apoB in the ethinyl estradiol-treated animal was apoBH. Nucleotide sequence analysis of hepatic apoB mRNA confirmed a marked decrease in the proportion of the apoBL mRNA in ethinyl estradiol-treated animals. After cessation of 17 alpha-ethinyl estradiol treatment, the hepatic apolipoprotein A-I synthesis rate, apolipoprotein A-I and A-IV mRNA levels, and the apoBH and apoBL synthesis rates, as well as plasma apolipoprotein and cholesterol levels, returned to normal. A major finding of the present study is that pharmacological doses of ethinyl estradiol do not affect total hepatic apoB synthesis, but increase the relative amount of apoBH synthesized.     

Boron estrogens: Synthesis,biochemical and biological testing of estrone and estradiol-17β 3-carboranylmethyl ethers

Synthesis, biochemical and biological testing of the first carborane derivatives of estrogens are described. Estrone 3-carboranylmethyl ether was synthesized in two steps from estrone. Reduction of estrone 3-carboranylmethyl ether with sodium borohydride provided estradiol-17β 3-carboranylmethyl ether. Enzyme kinetic measurements showed that estrone 3-carboranylmethyl ether is a substrate for human placental 17β-hydroxy-steroid dehydrogenase with Km = 5×10^?6M, and Vmax = 0.016 μmol min^?1 μg^?1. The relative affinity constant of estradiol-17β 3-carboranylmethyl ether for rat uterine estrogen receptor was 0.5 (compared with a value of 100 for estradiol-17β). Consistent with its low affinity for estrogen receptor, the dose-dependent uterotropic response to estradiol-17β 3-carboranylmethyl ether in castrated female rats was one sixtieth that of estradiol-17β. None of the tested rats had a toxic reaction to estradiol-17β 3-carboranylmethyl ether. These results demonstrate that exceptionally stable carborane derivatives of estrogens can be synthesized with preservation of their biochemical and biological properties. Boron-containing estrogens may be useful for thermal neutron capture therapy of cancers with estrogen receptors to concentrate boron in the cell nucleus.     

Chemical synthesis of two novel diaryl ether dimers of estradiol-17beta

We recently detected the formation of estradiol-17beta (estradiol) dimers, linked together through a diaryl ether bond between the C-3 phenolic oxygen of one estradiol molecule and the 2- or 4-position aromatic carbon of another estradiol, following incubations of [3H]estradiol with human liver microsomes or cytochrome p450 enzymes in the presence of NADPH. Using estradiol as the starting material, we designed a four-step method for the chemical synthesis of these two estrogen dimers with the Ullmann condensation reaction as a key step. Step 1: Synthesis of 2- or 4-bromoestradiol from estradiol. Step 2: Protection of the C-3 phenolic hydroxyl group of the 2- or 4-bromoestradiol. Step 3: The Ullmann condensation reaction between the phenol-protected bromoestradiol and the estradiol potassium salt under our modified reaction conditions (with a 41% product yield). Step 4: Removal of the C-3 benzyl group by catalytic hydrogenation. The chromatographic and various spectrometric properties of the two synthesized compounds were identical to those metabolically formed by human cytochrome p450 3A4.     

17Beta-hydroxy-11alpha-(3'-sulfanylpropyl)oxy-estra-1,3,5(10)- trien-3-yl sulfamate--a novel hapten structure: toward the development of a specific enzyme immunoassay (EIA) for estra-1,3,5(10)-triene-3-yl sulfamates.

The title compound 17 has been synthesized for the use as hapten in the development of a competitive enzyme immunoassay for estrogen sulfamates. The synthesis started from estradiol diacetate 2. Oxyfunctionalization at C-11 to give 11alpha-hydroxy steroid 8 was accomplished by hydroboration/alkaline hydrogen peroxide oxidation of the 9(11)-dehydro derivative 7, which was obtained from compound 2 via 9-hydroxylation with dimethyldioxirane. After transformation of compound 8 into the allyl ether 9, the side chain was thio-functionalized at the omega-position affording the thioate 11 in two steps. Selective silylether deprotection at position 3 followed by sulfamoylation gave the sulfamate 19, which in turn was demasked at position 17 and treated with sodium borohydride/aluminum chloride to liberate the side chain thiol. Alternatively, title compound 17 was synthesized via the disulfides 13-16. For the preparation of the immunogen the title compound 17 was coupled to bovine gamma globulin in a two-step procedure using an amine and thiol specific bifunctional crosslinker. The immunization of rabbits resulted in the formation of antibodies which clearly discriminated the sulfamoylated estrogens from the non-esterified estrogens. The use of a biotinylated hapten derivative as a tracer in combination with a streptavidin-peroxidase-tetramethylbenzidine based detection system allowed the measurement of estradiol 3-sulfamate (1) in the range of about 1 to 1000 pg/well.     

Synthesis of a precursor for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide

Starting from 11 beta-hydroxytestosterone, we achieved the synthesis of a strategic precursor, C-9 (11) unsaturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (9a), for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide. We optimized the reaction conditions for catalytic reduction employing hydrogen and subsequent base hydrolysis followed by purification on Amberlite XAD-2 resin to obtain the saturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.     

Metabolism of radioactive 17 beta-estradiol 3-methyl ether by humans.

A mixture of 2-3H and 4-14C-17beta-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 and 35 percent of the 3H was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulted from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4-14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.     

Effects of progesterone and estradiol-17 beta on uterine secretion of prostaglandin F2 alpha in response to oxytocin in ovariectomized ewes

Twenty ovariectomized ewes were used in an experiment designed to examine the interaction of progesterone, estradiol, and oxytocin in the regulation of uterine secretion of prostaglandin F2 alpha (PGF2 alpha). All ewes underwent a steroid pretreatment that mimicked the changes in progesterone and estradiol which occur during the six days immediately prior to estrus. After pretreatment, ewes were randomly assigned to 1 of 4 treatment groups: 1) control (n = 4); 2) estradiol-17 beta (n = 6); 3) progesterone (n = 4); and 4) progesterone and estradiol-17 beta (n = 6). Progesterone was injected twice daily for 15 days. The dose of progesterone varied with day postestrus in a manner designed to simulate endogenous luteal secretion of progesterone. Estradiol-17 beta was administered in s.c. Silastic implants. The implants maintained circulating concentrations of estradiol at 3 pg/ml. On Days 5, 10, and 15 of treatment, ewes were injected with oxytocin (10 IU in 1.0 ml saline, i.v.). Jugular venous blood samples were collected beginning one-half hour prior to and continuing for 2 hours post-oxytocin injection for quantification of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM). No changes in concentration of PGFM following injection of oxytocin were observed on Day 5 or 10 in any treatment group. Concentrations of PGFM increased following injection of oxytocin on Day 15 only in groups receiving progesterone. Both the area under the PGFM response curve (p = 0.08) and peak response (p = 0.06) were greater in ewes treated with progesterone and estradiol-17 beta than in those receiving progesterone alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive protein-binding assay of estrone, estradiol-17 or estradiol-17 in human or ruminant plasma

A procedure for the assay of estrone, estradiol-17β or estradiol-17α in plasma is described. The technique also appears applicable to the assay of estriol in plasma. The procedure uses a semi-automatic extraction of plasma, rapid micro-alumina column chromatography and competitive binding of the estrogens to stable proteins of sheep uterine cytosol. The use of alumina column chromatography results in consistently low blanks. The assay has been evaluated for the measurement of estradiol-17β and estrone in human and sheep plasma, and for estradiol-17α and estrone in goat plasma. The change in binding affinity of estradiol-17α relative to estradiol-17β when incubated in sheep uterine cytosol at two different temperatures (25°C and 4°C), makes it possible to differentiate the two epimers of estradiol. Measurement of estradiol-17β down to 10 pg and of estrone and estradiol-17α to 25 pg are maintained in routine analyses. The specificity of the procedure was thoroughly checked by various methods, including comparison with spectrophotofluorimetric analysis.     

Release of prostaglandin F, regression of corpora lutea and induction of premature parturition in goats treated with estradiol-17β

Premature parturition was induced in five pregnant goats infused intravenously with 4.65–8.4 mg estradiol-17β but not in one treated with 5.85 mg estradiol-17α. A single intramuscular injection of 12 mg estradiol benzoate (8.8 mg estradiol-17β equivalents) was also effective. These doses were estimated to provide plasma concentrations of estradiol-17β in the physiological range for animals at spontaneous parturition. Circulating plasma concentrations of progesterone decreased and lactogenesis occurred before all instances of induced parturition but no such changes resulted from infusion of estradiol-17α. Placental delivery was normal in all animals but neonates of more than 10 days prematurity were non-viable.In three of the five goats infused with estradiol-17β, evidence was obtained for release of F-group prostaglandins from the uterus at the time of onset of the decline in progesterone.     

Structure-activity relationships of estrogens. Effects of 14-dehydrogenation and axial methyl groups at C-7, C-9 and C-11

Thirty compounds were evaluated in the rat for uterotropic effects, inhibition of gonadotropin release, and competitive displacement of (3H) estradiol-17 beta from uterine cytosolic preparations. 7 alpha-Methylestradiol-17 beta was 150% as active as estradiol-17 beta as an uterotropic agent. Estradiol-17 beta was the most active inhibitor of gonadotropin release. 11 beta-Methylestradiol-17 beta had 124% of the activity of estradiol-17 beta in displacing (3H) estradiol-17 beta from the "estrogen receptor." The 9 alpha-methyl group considerably decreased the potency of estrogens in any of the three assays. The 14-dehydro modification was advantageous only in the estradiol-17 beta 3-methyl ether series. Uterotropic activities and inhibition of gonadotropin release did not parallel. The best compound for inhibiting gonadotropin release, as compared to uterotropic activity, was estrone. The "estrogen receptor" assay data correlated fairly well with uterotropic assay data, but only for compounds having free 3-hydroxyl groups; even so, some exceptions were noted.     

Metabolism of norethynodre, a 19-nor progestin: subcellular localization of enzyme activity

The subcellular distribution of the enzymes involved in the metabolism of norethynodrel (17 alpha-ethynyl-17 beta-hydroxy-estr-5(10)-en-3-one) to the 3alpha and 3beta diols (17 alpha-ethynyl-3alpha (or 3beta-17 beta-dihydroxy-estr-5(10)-ene) and 17 alpha-ethinyl estradiol was studied. The purity of the male rat liver subcellular fractions was evaluated by the use of marker enzymes. Sample sections were viewed by electron microscopy. The data showed that the cytosol fraction contained the highest relative specific activity for the hydroxysteroid dehydrogenases required for the formation of the diols. The cytosol fraction also contained the highest total activity. The enzymes required for the formation of ethinyl estradiol were distributed equally among mitochondrial and microsomal fractions, however, the highest relative specific activity was associated with the heavy microsomal fraction (18,000 g).     

17 alpha-substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.     

Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver

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.