Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

³H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the $\text{Torpedo}$ fish. The K_d for binding to intact and solubilized rat brain membranes was 5.6 × 10^?9 M and 1.1 × 10^?8M respectively, and the binding capacity 2.0 × 10^?14 and 3.0 × 10^?13 moles /mg protein respectively. The K_d for Torpedo membranes was 3.1 × 10^?7M and the binding capacity 6.8 × 10^?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in $\text{Torpedo}$ membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with ³H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.     

Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 10¹⁰ sites per 100 μg protein in the youngest group (Pool A) to 46 × 10¹⁰ sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ (“empty site”), was 1.53 × 10⁸M^?1 in Pool A and was only slightly higher than $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ (“filled site”). $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ was higher in Pool B, 1.75 × 10⁸M^?1, and in Pool C reached a value of 5.63 × 10⁸M^?1. In Pool C $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ was 2.3 × 10⁸M^?1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 10¹⁰ sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 10¹⁰). In addition, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ increased from 0.75 × 10⁸M^?1 at 14 days to 3.02 × 10⁸M^?1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.     

Characterization of oxytocin receptors in the uterus and mammary gland.

High affinity binding sites for 3[H] oxytocin have been demonstrated in particulate fractions from rat uterus and oviduct, myometrium from the sow, ewe and human, ewe endometrium, and mammary gland from the lactating rat. The binding activity has been localized to enriched plasma membrane fractions from the rat uterus and mammary gland; cells isolated from the mammary gland also bind oxytocin. The apparent dissociation constant (Kd) for the interaction of oxytocin with its binding sites in a variety of tissue preparations is in the nanomolar range. The concentration of oxytocin eliciting half-maximal contraction of the rat isolated uterus corresponds to the apparent Kd of oxytocin interaction with uterine particulate fractions. Binding is specific with respect to the target tissue or cell, as well as to the ligand. The affinity of binding sites for oxytocin analogues corresponds generally to their potencies as agonists or antagonists. Factors that affect the binding of oxytocin affect the biological response in the same way. For example, certain divalent metal ions, which increase oxytocin binding activity, enhance the sensitivity of the contractile response of the uterus and mammary gland to oxytocin. Estrogen administration, which increases the uterine binding of oxytocin, increases the sensitivity of the myometrium to oxytocin. The myometrium binds the most oxytocin at estrus and is most sensitive to oxtocin at that time. The dgree of stimulation by oxytocin of prostaglandin F2alpha synthesis by ewe endometrium is paralleled by an increased concentration of oxytocin binding sites. The marked increase in sensitivity to oxytocin of the rat uterus occurring on the day of parturition also is reflected by the amount of oxytocin bound by the uterus. Because of the many correlations between oxytocin binding and bioactivity, it appears that oxytocin binding sites on the plasma membrane of target cells constitute the recognition part of oxytocin receptors.     

Receptor-like stereospecific binding of a pyrethroid insecticide to mouse brain membranes

A heterogeneous particulate fraction of mouse brain homogenates binds NRDC 157 (3-phenoxybenzyl [1$\text{R}$,$\text{cis}$]-3-(2,2-dibromovinyl)-2,2- dimethylcyclopropanecarboxylate), a potent pyrethroid insecticide, stereospecifically and with high affinity. Stereospecific binding is a minor component of total binding (2.8%); the remainder of observed binding is predominantly nonspecific and unsaturable. Stereospecific binding is half-saturated at 4×10^?8$\text{M}$ and fully saturated at concentrations in excess of 1×10^?7$\text{M}$. The stereospecific binding capacity of this preparation was 200–250 pmoles of NRDC 157 per gram equivalent of brain tissue (2.3–2.8 pmol/mg protein). This binding site may represent the neural receptor involved in the stereospecific toxic action of pyrethroids.     

Binding of opiates and endogenous opioid peptides to neuroleptic receptor sites in the corpus striatum.

Some opiates with morphinan- and benzomorphan-structures possess affinities for neuroleptic receptors as revealed by their abilities to compete with ³H-spiroperidol for common binding sites in rat striatum $\text{in vitro}$ (IC₅₀ in the range between 10^?6 and 10^?5M). The binding of these opiates to neuroleptic receptors appears to be of pharmacological significance, since $\text{in vivo}$ studies in mice revealed a small but significant displacement of spiroperidol by high doses of the opiate antagonist levallorphan from specific binding sites in the striatum. In addition, there exists some correlation between the ability of opiates to bind to neuroleptic receptor sites $\text{in vitro}$ and their potency to evoke “bizarre behavior” in rats $\text{in vivo}$. In contrast, a wide variety of other opiates having morphine-, morphinone- or oripavine-structure showed no affinity for neuroleptic binding sites $\text{in vitro}$ (IC₅₀ greater than 10^?4 M). Of the opioid peptides (methionine-enkephalin, leucine-enkephalin and β-endorphin) none has an affinity for neuroleptic binding sites. A variety of other peptides were also investigated but did not interfere with spiroperidol binding. Only ACTH showed a moderate affinity for neuroleptic binding sites.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Properties of α-bungarotoxin binding sites in foetal human brain

Maximum levels of binding of α-bungarotoxin to foetal human brain membranes were found to remain essentially constant at 30–50 fmol/mg protein (1.1–1.5 pmol/g wet weight in whole brain) between gestational ages of 10 and 24 weeks. Equilibrium binding of α-bungarotoxin to both membranes and to detergent extracts showed saturable specific binding to a single class of sites with K_d (app) values of 3.5 × 10^?9 M and 2.4 × 10^?9 M respectively. Association rate constants, determined from time courses of binding of α-bungarotoxin to membranes and detergent extracts, were 2.3 × 10⁵ M^?1 sec^?1 and 2.6 × 10⁵ M^?1 sec^?1 respectively. Dissociation of α-bungarotoxin from both membrane and detergent extracts showed a rapid initial rate with $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx 15 min which, in the case of the detergent extract, was followed by a slower dissociation accounting for the remaining 20% of the bound ligand. Competition studies with a number of cholinergic ligands indicated that the α-bungarotoxin-binding sites in foetal brain display a predominantly nicotinic profile.     

Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells

We have previously shown that gonadotropin releasing hormone (GnRH) and its agonists inhibit ovarian functions by a direct action on ovarian granulosa cells $\text{in vitro}$. A labeled GnRH agonist, [des-Gly¹⁰, D-Ser (TBu)⁶, Pro⁹-NHEt]GnRH, was used here to examine the possibility that these inhibitory actions of GnRH were mediated through specific receptors which recognize GnRH. Ovarian membrane fractions obtained from immature, hypophysectomized diethylstilbesterol-treated rats were incubated with the ¹²⁵I-GnRH agonist and specific binding was determined by a filtration assay. Stereospecific, high affinity binding was detected in the ovarian membranes; the dissociation constant for the labeled GnRH agonist was determined to be 0.84 ± 0.33 × 10^?10 M and the binding capacity was calculated to be 12.9 fmol/mg protein, or 0.142 fmol/μg DNA. The binding affinity for the GnRH decapeptide was 3.3 times lower than that of the GnRH agonist whereas two GnRH partial peptides did not compete for the ¹²⁵I-agonist binding. After sequential treatment with FSH, LH and prolactin to the hypophysectomized female rats, the ovarian GnRH binding capacity increased per ovary, but decreased per mg ovarian protein.Furthermore, ovarian granulosa cells were isolated and their binding capacity was determined to be 25.2 fmol/mg protein, or 0.133 fmol/μg DNA, suggesting that the granulosa cells contain GnRH binding sites. Thus, this report demonstrates the presence of stereospecific, high affinity GnRH binding sites in the rat ovarian granulosa cells.     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Binding studies of Mn+2 to isolated acetylcholine receptor as a probe for cation-receptor interaction.

The paramagnetic cation Mn⁺² binds to $\text{Torpedo californica}$ acetylcholine receptor (AcChR) at sites with at least two different affinity constants. For each α-Bungarotoxin (α-Bgt) binding site AcChR has between 3 to 4 Mn⁺² sites with Kd values of 1.74 ± 1.0 × 10^?4 M. An additional 10–12 sites/α-Bgt site have a weaker affinity for Mn⁺² (Kd ? 1 mM). The α-Bgt does not displace bound Mn⁺², however Ca⁺² displaces all bound Mn⁺² in a competitive fashion with Kd of 0.90 × 10^?3 M and Mg⁺² is as effective as Ca⁺² in the displacement. Decamethonium, carbamylcholine and NaCl at high concentrations are also effective in displacing Mn⁺². A constant enhancement value (?_b) for the binary metal · AcChR complexes was obtained when simultaneous EPR measurements and the water proton relaxation rates were made. Similarity of the AcChR environment and/or coordination number for the Mn⁺² sites in AcChR is inferred. It appears that Mn⁺² binds to many AcChR sites, different from those responsible for binding cholinergic ligands. The Mn⁺² site seem to be the same as those responsible for binding the electrophysiologically significant Ca⁺².     

In vitro triiodothyronine binding to non-histone proteins from rat liver nuclei

$\text{In vitro}$ incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 10⁷ M^?1 h^?1 and the rate constant of dissociation was 0.015 h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10^?9 M. The maximum binding capacity was 10^?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone.     

Properties of [3H] diazepam binding to rat peritoneal mast cells

Benzodiazepine binding to rat peritoneal mast cells was investigated using [³H] diazepam as the radioactive probe. The specific binding of [³H] diazepam reaches equilibrium within 10–15 min, is saturable and is linear with cell number. Scatchard analysis of equilibrium binding indicates the existence of only one class of binding sites with a K_D = 90 ± 10 nM and B_max of 261 ± 60 fmoles/10⁶ cells. The binding of [³H] diazepam is temperature dependent, the highest amount is bound at 0°C and shows a pH-optimum between pH 6.8 – 7.4. The binding of [³H] diazepam is reversible with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2 ± 0.2}\text{min.}$ Based on the relative potency of clonazepam and Ro5-4864 in displacing the specific [³H] diazepam binding, the binding sites in the mast cell are similar to those in the peripheral tissues like lung, liver, and kidney and are different from those in the brain. These data indicate that the mast cells have benzodiazepine binding sites which are of the peripheral type.     

Differential effects of sodium on two types of opiate binding sites.

Effects of Na⁺ on saturable naloxone binding were studied $\text{in vitro}$ using particulate fractions obtained from rat brain homogenates. Na⁺ stimulated the saturable naloxone binding in thalamus-hypothalamus regions, but inhibited it in cerebellum. These findings strongly support the hypothesis that two types of naloxone binding sites exist in brain tissues.     

D-LSD binding to brain homogenates: possible relationship to serotonin receptors

Using a rapid filtration technique we find stereospecific high-affinity $\text{d}$-LSD binding (4 × 10^?9M, half-saturation) in brain fractions from a number of subcortical as well as cortical brain regions. Among the putative neurotransmitters tested only serotonin effectively displaces $\text{d}$-LSD from this specific binding site. Moreover, only the serotonin-displaceable component of binding is saturable in a high-affinity range. No change is observed in specific $\text{d}$-LSD binding in forebrain homogenates from rats in which ascending serotonergic pathways are destroyed by lesions in the raphe nucleus. We conclude that a vast majority of the $\text{d}$-LSD binding sites may be associated with a postsynaptic serotonin receptor rather than a presynaptic receptor associated with serotonergic (raphe) inputs.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Demonstration of specific "high affinity" binding sites for [3H] imipramine on human platelets

High affinity and saturable binding sites for [³H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [³H] imipramine binding. The binding of [³H] imipramine to human platelets is of high affinity $\text{(}\text{Kd}\text{? 1.4}\text{nM}\text{)}$, saturable $\text{(}\text{Bmax}\text{? 625}\text{fmols/mg prot}\text{)}$, and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [³H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.     

Morphine release and displacement by naloxone in vivo in morphine naive and withdrawn rats

Male Long-Evans rats, implanted in the lateral cerebroventricle with chronic indwelling push-pull cannulae, were perfused (10 μl/min) for 120 min: 20 min with 1.5 × 10^?6M morphine in sterile isotonic saline containing 2.3 mM CaCl₂ (vehicle); 40 min with vehicle; 20 min with 1.5 × 10^?6M morphine; 10 min with vehicle and 30 min with 1 × 10^?6M naloxone in vehicle. These rats and drug-naive rats were implanted s.c. with 2 × 50 mg morphine pellets. After 72 hr the pellets were removed and 18–24 hr later the above perfusion procedure was repeated. The amount of morphine collected in the perfusate during the washout with naloxone was elevated, compared to the amount collected during the corresponding time of the washout with vehicle for both naive and withdrawn groups. The enhanced morphine release during the washout with naloxone did not differ significantly between the naive and withdrawn rats. However, significantly less morphine was recovered in the perfusate collected during the vehicle washout from the withdrawn rats, compared to that collected from the naive rats. The data suggest that $\text{in vivo}$ morphine is specifically bound to receptors and is sensitive to naloxone displacement. It is also concluded that morphine is differentially taken up or otherwise disposed of by brains of rats which are in opiate withdrawal.     

Kinetic properties of rat hepatic prolactin receptors

Binding of ¹²⁵I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent K_d of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K₁, of 2.1 × 10⁷, 1.4 × 10⁷, 1.2 × 10⁷ and 4 × 10⁶ M^?1. min^?1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K_?1=1.26 × 10^?2. min^?1 ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=55}\text{minutes}$) and a slower phase with K_?1=1.103 × 10^?3. min^?1. The apparent K_d (from K_?1K₁) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone.