High Affinity Binding Capacity Research Articles

Turnover of Functional Basic Fibroblast Growth Factor Receptors on the Surface of BHK and NIH 3T3 Cells

The recovery of functional cell-surface bFGF receptors after trypsin treatment was studied in BHK cells and NIH 3T3 cells. Restoration of functional bFGF receptors occurred at an approximately linear rate with 50% of the high-affinity binding capacity restored after 4 hr. Restoration of functional receptors required protein synthesis but not RNA synthesis. Upon exposure of BHK cells to bFGF, cell-surface receptors were rapidly lost, with only 25% remaining after 1 hr. When the bFGF was removed, down-regulated BHK and NIH 3T3 cells recovered cell-surface receptors at about the same rate observed in trypsin-treated cells. The recovery of receptors after down-regulation was inhibited by protein synthesis inhibitors. Addition of the protein synthesis inhibitor cycloheximide to unperturbed cultures of BHK or NIH 3T3 cells resulted in a time-dependent loss of cell-surface bFGF receptors, demonstrating that the receptors turn over constantly in the absence of ligand. These results suggest that bFGF receptors do not recycle and must be continuously synthesized.

Solubilization of High Affinity Corticotropin-Releasing Factor Receptors from Rat Brain: Characterization of an Active Digitonin-Solubilized Receptor Complex

The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.

GnRH RECEPTORS IN HUMAN GRANULOSA CELLS: ANATOMICAL LOCALIZATION AND CHARACTERIZATION BY AUTORADIOGRAPHIC STUDY

The presence of receptors for GnRH in human ovary has been investigated by quantitative autoradiography. Simultaneous visualization and characterization of specific receptors on frozen sections were obtained on six pairs of human ovaries. Among them only one exhibited a large preovulatory follicle. This dominant follicle exhibited a specific and high affinity binding capacity for 125I-GnRHa exclusively localized on the granulosa cell layer. Analysis of saturation curve indicates a Kd value of 0.22 nM and Bmax of 9.6 fmol/mg protein. In contrast LH-hCG binding sites were present in all antral follicles. These data demonstrate for the first time the presence of high affinity GnRH receptors in human granulosa cells at a late stage of follicular maturation.

Decrease of prostaglandin I2 binding sites in thyroid cancer

The properties of specific prostaglandin I2 (prostacyclin, PGI2) binding sites in normal thyroid tissue have been characterised. Tissue samples obtained intraoperatively from patients with 'cold' solitary thyroid nodules (as preoperatively selected by thyroid gland scintigraphy, thyroid gland ultrasonography and Papanicolaou cytology following fine needle aspiration of the nodule area) have been used for thyroid membrane preparation. Employing [3H]iloprost, a chemically stable PGI2-analogue as a radioligand, saturation experiments for comparative binding studies have been attempted. Scatchard analysis of the binding data obtained for normal thyroid parenchyma distant to the nodule area revealed heterogeneity of the [3H]iloprost sites exhibiting a high-affinity binding capacity (Bmax) of 613.2 +/- 130.4 fmol mg-1 membrane protein and a low-affinity binding capacity of 5.1 +/- 1.6 pmol mg-1 membrane protein. The equilibrium dissociation constant (Kd) amounted to 18.9 +/- 8.9 nM and to 131.5 +/- 39.2 nM, respectively. Scatchard analysis of the binding data obtained for benign thyroid adenoma indicated significant lower binding capacities exhibiting a Bmax of 325.8 +/- 110.0 fmol mg-1 membrane protein (Kd: 31.0 +/- 7.5 nM) for the high-affinity sites and of 3.9 +/- 2.5 pmol mg-1 membrane protein (Kd: 364.9 +/- 183.6) for the low affinity sites. In cancer tissue a selective loss of the low affinity sites and a significant diminution of the high-affinity sites was observed: in well differentiated cancer the high-affinity sites showed a Bmax of 299.7 +/- 46.0 fmol mg-1 membrane protein (Kd: 38.9 +/- 7.3 nM), in anaplastic cancer, less differentiated papillar and follicular cancers of 180.6 +/- 25.1 fmol mg-1 membrane protein (Kd: 54.6 +/- 16.7 nM). Well differentiated papillar and follicular cancers did not differ from each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacology and molecular identification of vasoactive intestinal peptide (VIP) receptors in normal and cancerous gastric mucosa in man

In human antral membranes, VIP and its natural analogs inhibited the binding of HPLC-purified 125I-VIP, according to the following order of potency: VIP greater than rh GRF greater than helodermin greater than r PHI greater than PHM greater than p PHI greater than hp GRF greater than h, p secretin. No specific binding was detected in plasma membranes purified from the human fundus. In human antral membranes, Scatchard plots were compatible with the existence of two classes of VIP receptors, the first class with high affinity and low binding capacity (Kd = 0.1 nM, Bmax = 10 fmol/mg protein) and another class with a low affinity and higher binding capacity (Kd = 12) nM, Bmax = 1,000 fmol/mg protein). The structure of the VIP receptor in purified plasma membranes prepared from human antral glands and from the HGT-1 human gastric cancer cells was subsequently probed using the cross-linking reagent DSP and 125I-VIP. In agreement with the pharmacological study and the Scatchard analysis of the binding data, SDS gel electrophoresis of the solubilized receptor identified two radiolabeled peptides Mr 67,000 and 34,000 containing disulfide bonds. According to its sensitivity to low doses of VIP and to GTP, the Mr 67,000 binding site represents the membrane domains involved in the physiologial regulation of adenylate cyclase by VIP in normal and transformed human gastric epithelia.

Influence of lipid environment on insulin binding in cultured hepatoma cells

The influence of alterations of plasma membrane physico-chemical properties on insulin binding have been characterized in an insulin-sensitive rat hepatoma cell line adapted to grow for several generations in culture medium enriched with linoleic acid (18:2) or with 25-hydroxycholesterol. The cells took up 18:2 and 25-hydroxycholesterol added to the culture medium, without exhibiting any sign of intolerance or intoxication. These compounds respectively increased and decreased membrane fluidity at 37°C. The cells demonstrated extensive changes in insulin binding parameters in response to experimental modifications of their membrane lipid composition. When determined at 4°C, insulin receptors were present in the control cells at 136 000 sites/cell but this fell to 111 000 ( P < 0.05) in cells enriched in 18:2, and rose to 176 000 ( P < 0.001) in hydroxysterol-grown cells. According to a two-site model, the main effect of 18:2 was a significant increase of the number of high-affinity sites with a concomitant decrease of low-affinity sites. The hydroxysterol had the opposite effects on these parameters. The high-affinity insulin binding capacity of the hepatoma cells was affected by lipid supplementation in a similar way, whether it was determined at 4°C or at 37°C. Assuming a negative cooperativity model, 18:2 enhanced the degree of negative cooperativity among the sites, while 25-hydroxycholesterol reduced it. The time-course of insulin-induced receptor down-regulation was accelerated in the cells enriched in polyunsaturated fatty acids, but reduced in cells exposed to 25-hydroxycholesterol. These insulin-binding alterations cannot be directly related to modifications of cellular growth rate, receptor internalization or membrane fluidity per se, and are discussed as being more likely due to membrane lipid composition than to overall cell metabolism modifications.

Low-density lipoprotein receptor activity in the guinea pig adrenal cortex. II. Zonal response to aminoglutethimide and 17α-ethinylestradiol

Low-density lipoprotein (LDL) receptor activity and the concentration of cholesterol were measured in the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig to examine the relation between cholesterol content and LDL receptor activity. While the concentration of cholesterol was 2–3-times higher in the outer cortical zone, the maximum high-affinity binding capacity for LDL was essentially the same for the two zones, or slightly higher for the inner zone. Adrenocorticotrophic hormone (ACTH) caused a significant increase in LDL receptor activity only in the outer zone, but led to a reduction in the cholesterol content in both adrenocortical zones. The treatment of animals with 17α-ethinylstradiol also resulted in a reduction of cholesterol in both adrenocortical zones, but an increase in LDL receptor number only in the outer zone. The latter effect was partially reversed by the administration of dexamethasone. Aminoglutethimide, which was used in a dose that did not block steroidogenesis but did block the hydrolysis of cholesteryl esters in response to ACTH, did not prevent the ACTH-induced increase in LDL receptor number in the outer zone. Thus, the number of LDL receptors was increased in the zona fasciculata by ACTH in the absence of a reduction in cellular cholesterol content, while the number of LDL receptors in the zona reticularis was not increased by ACTH even in the face of a reduction in cellular cholesterol. Exclusive of the experiments employing aminoglutethimide, when the cellular cholesterol content was plotted against LDL binding activity, an excellent inverse correlation was revealed for the zona fasciculata, but essentially no correlation was noted for the zona reticularis. It is concluded that the outer and inner cortical zones of the guinea pig adrenal are quite distinct in the nature of their LDL receptor activity and regulation: the LDL receptor of the outer zone appears to function in a way similar to what has been reported for the whole adrenal cortex of other species in that receptor number correlates with tissue cholesterol content and is primarily regulated by ACTH; the LDL receptor number of the inner zone, however, does not correlate with tissue cholesterol content and is apparently not regulated by ACTH.

Low Density Lipoprotein Receptor Activity in the Guinea Pig Adrenal Cortex. I. Zonal Characterization and Response to Adrenocorticotropin

The binding of [125I]iodo-low density lipoprotein ([125I]iodo-LDL) to isolated membranes was determined for the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the guinea pig adrenal cortex. Binding of [125I]iodo-LDL to membranes from both zones was found to be highly specific and dependent on the presence of divalent cations, such as calcium. Saturation analysis revealed that the maximum high affinity binding capacity was similar for both zones under control conditions (approximately 400 ng/mg protein). When ACTH was administered, however, the maximum high affinity binding capacity increased in the outer zone by at least 3-fold, while there was no significant change for the inner zone. It was also noted that dexamethasone administration decreased [125I]iodo-LDL high affinity binding activity to less than half the control level in the outer zone without altering the binding activity in the inner zone. The cholesterol content in the outer zone was 2-3 times greater than that in the inner zone. When ACTH was administered, the concentration of cholesterol decreased significantly in both zones. Thus, ACTH appeared to influence cholesterol metabolism in the outer and inner adrenocortical zones, but was able to modulate LDL receptor activity only in the outer zone.

Characterization of insulin receptors in isolated epithelial cells of rat ventral prostate: effect of fasting.

Insulin receptors have been characterized in rat prostatic epithelial cells by using [125I]insulin and a variety of physicochemical conditions. The binding data at equilibrium (2 h at 15 degrees C) could be interpreted in terms of two populations of insulin receptors: a class of receptors with high affinity (Kd = 2.16 nM) and low binding capacity (28.0 fmol mg-1 protein), and another class of receptors with low affinity (Kd = 0.29 microM) and high binding capacity (1.43 pmol mg-1 protein). Proinsulin exhibited a 63-fold lower affinity than insulin for binding sites whereas unrelated peptides were ineffective. The specific binding of insulin increased by about 50 per cent after 96 h of fasting; this increase could be explained by an increase of both the number of the high affinity-low capacity sites and the affinity of the low affinity-high capacity sites. These results together with previous studies on insulin action at the prostatic level strongly suggest that insulin may exert a physiological role on the prostatic epithelium.

Characterization of a [ 3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17β-hydroxy-17α-methyl-estra-4, 9, 11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [ 3H] R 1881 macromolecular complex which sediments in the 8–9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [ 3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [ 3H] R1881 was recovered by thin layer chromatography. Characteristics of this [ 3H] R1881-8S binding protein include high affinity ( K d = 2.3 ± 41 nM) and low binding capacity (18.8 ± 3.3 fmol/mg cytosol protein), precipitability in 0–33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [ 3h] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a K d = 2.8 nM for [ 3H] R1881, and is androgen specific (testosterone > 5α-dihydrotestosterone > estradiol > progesterone > cyproterone acetate > di-ethylstilbestrol > dexamethasone > triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.

Vip binding to epithelial cell membranes of rat ventral prostate: Effect of guanine nucleotides

1. 1.|Specific binding of vasoactive intestinal peptide (VIP) to epithelial cell membranes of rat ventral prostate was reversible, saturable and dependent on time and temperature. 2. 2.|The data suggested the presence of two classes of VIP receptors: a class with high affinity ( K d = 1.7 nM) and low binding capacity (0.5 pmol VIP/mg protein), and another class with low affinity ( K d = 36.2 nM) and high binding capacity (7.5 pmol VIP/mg protein). 3. 3.|Chicken VIP and porcine secretin recognized VIP receptors but exhibited a 10-fold higher and a 40-fold lower affinity than porcine VIP, respectively. However, glucagon, somatostatin, Met-enkephalin and cholecystokinin were ineffective. 4. 4.|GTP inhibited markedly the interaction of VIP with membranes by increasing the rate of dissociation of peptide bound to its receptors. GDP and Gpp(NH)p behaved as GTP but other purine nucleotides and nucleosides did not show any effect.

The mitochondrial uncoupling protein from guinea-pig brown adipose tissue. Synchronous increase in structural and functional parameters during cold-adaptation.

The time-course for the induction of the uncoupling pathway in the inner membrane of brown-fat mitochondria from cold-adapting guinea pigs was studied. The amount of the protein was quantified from the capacity for high-affinity binding of GDP to the intact mitochondria, and was compared with the functional parameters diagnostic of the protein, namely the nucleotide-sensitive proton conductance and the sensitivity to uncoupling by low concentrations of fatty acids. A monophasic increase in nucleotide titre was observed, with no evidence of an early 'unmasking' of preexisting nucleotide-binding sites. The nucleotide-sensitive conductance increased in precise synchrony with the nucleotide-binding capacity. Mitochondria from newborn animals, and those from acutely cold-adapted animals, showed anomalously low sensitivities to uncoupling by fatty acids.

Properties of the Mg 2+-induced low-affinity nucleotide binding site of (Na ++ K +)-activated ATPase

(1) The Mg 2+-induced low-affinity nucleotide binding by (Na + + K +)-ATPase has been further investigated. Both heat treatment (50–65°C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the K d value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na + + K +)-ATPase activity. (2) Thermodynamic parameters (ΔH° and ΔS°) for the apparent affinity in the ATPase reaction ( K m ATP) and for the true affinity in the binding of Ado PP[NH] P ( K d and K i) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the K m value, which thus is smaller than the K d value. (3) Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the K d value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and Ado PP[NH] P and least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the K d value. (4) The nucleotide specificity of low-affinity binding site is the same for binding (competition with Ado PP[NH] P) and for the ATPase activity (competition with ATP): Ado PP[NH] P > ATP > ADP > AMP. (5) The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the K d value is not increased more than by ouabain alone. (6) It is inferred that the low-affinity site is Iocated on the enzyme, more specifically its α-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.

Endocrine aging in C57 BL mice—II. Dynamics of estrogen receptors in the hypothalamic-pituitary axis

Specific cytosolic and nuclear binding sites for estrogens were measured in the hypothalamic-pituitary axis (HPA) of young (4–8 months) and old (16–18 months) C57 BL mice in order to determine any age-related alteration in hormone-receptor interaction. Our results indicated no age differences in the affinity ( K D = 0.89 ± 0.03 (SEM) vs 1.09 ± 0.2 × 10 −9 M), the specificity, the sedimentation profile (6 s) or in the number (98.9 ± 4.9 vs 84.4 ± 2.3 fmol/mg protein) of unoccupied estrogen binding sites in the cytosols. Estradiol administration to young mice induced a complete translocation of cytosolic estrogen receptors to the nucleus, and two types of nuclear binding sites were observed: Type I were specific for estrogens with high affinity ( K D = 0.51 ± 0.06 × 10 −9 M) and low binding capacity (115.1 ± 22.7 fmol/mg DNA) and sedimented in the 4.0 s area, while Type II binding sites showed a much higher capacity and lower affinity for R2858. HPA nuclear suspensions of aged untreated mice showed undetectable ( < 50 fmol/mg DNA) levels of nuclear estrogen receptors and E 2 pre-treatment resulted in a significant increase in both types of binding sites. While no significant changes in the physicochemical characteristics of these nuclear receptors were observed, when compared to young animals, aging was manifested by a transiocation defect in the HPA of C57 BL mice. These results suggest aging changes in the endocrine regulating centers of the brain with defective activation of estrogen receptors.

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that [formula omitted]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist (±)-[ 3 H]hydroxybenzylisoproterenol to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k 1=1.37·10 7·M −1·min −1). Dissociation of specific binding by 0.5 mM (−)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k 2 of 0.106·min −1 and 0.011·min −1.[ 3H]Hydroxybenzylisoproterenol binding was saturable ( B max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [ 3H]hydroxybenzylisoproterenol binding sites, one having high affinity ( K D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity ( K D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [ 3H]hydroxybenzylisoproterenol binding with the following order of potency=(−)-propranolol>(−)-isoproterenol>(−)-norepinephrine≈ (−)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β 1-adrenergic receptors. Competition curves of [ 3H]hydroxybenzylisoproterenol binding by the β-agonist (−)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (−)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [ 3H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (−)-isoproterenol, then number of [ 3H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (−)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (−)-isoproterenol to displace [ 3H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [ 3H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [ 3H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [ 3H]dihydroalprenolol in these cells.

Catecholamine binding to the α-adrenergic receptors of hamster adipocytes evidence that guanine nucleotides regulate this binding to the α2-receptor subtype

The binding characteristics of the α-component of (−)-[ 3H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, reaching equilibrium in 20 min at 25°C. Dissociation of specific binding by 10 μM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [ 3H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity ( D = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity ( K D = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands of competed with [ 3H]norepinephrine binding in the following order of potency: (−)-norepinephrine>(−)-epinephrine>>(+)-norepinephrine>(−)-isoproterenol. Displacement by the selective α-adrenergic drugs prazosin, clonidine and yohimbine yielded biphasic curves consistent with binding of [ 3H]norepinephrine to both α 1- (14–22%) and α 2- (78–86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [ 3H]dihydroergocryptine, it severely reduced the binding affinity of (−)-epinephrine, (−)-norepinephrine and the selective α 2-agonist, clonidine. The inhibitory effects of clonidine and of the α-component of (−)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [ 3H]norepinephrine and [ 3H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the α-inhibitory effect of norepinephrine on cyclic AMP production. These data led to concluded that [ 3H]norepinephrine can be successfully used to study the entire α-adrenergic receptor population of hamster fat cells and that the predominant α 2 -receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.

Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops).

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for 125 I-hCG, (0.9-2.5 × 109 M-1 , and 0.7-1.64 × 109 M-1 respectively, for green and rhesus monkeys) and low binding capacity (0.343-0.682 fmol/mg and 0.198-0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 109 M-1 ) and low binding capacity (1.04 fmol/mg tissue homogenate) for 125 I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.

Apparent “negative cooperativity” kinetics in the absence of a nonlinear Scatchard plot of thyrotropin-receptor interaction in a human thyroid adenoma

A human thyroid adenoma (benign nodule) was identified which exhibited a linear Scatchard plot of 125I-TSH binding, characteristic of a single class of binding site with high affinity (K d = 0.5±0.1 nM) and low binding capacity (0.8±0.2 pmol/mg protein). In contrast, Scatchard analysis of binding to adjacent normal thyroid was nonlinear, suggesting the presence of high and low-affinity binding sites with K d's of 0.4±0.2 and of 27.9±11.0 nM and capacities of 0.7±0.3 and 1.8±1.0 pmol/mg protein, respectively. Dissociation of bound 125I-TSH from membranes of both adenoma and normal tissue revealed identical enhancement of dissociation in the presence of excess native hormone, thought to be evidence for the “negative cooperativity” model of hormone-receptor interaction. Furthermore, adenylate cyclase from both tissues was equally responsive to TSH. Thus, a thyroid adenoma which contains TSH-responsive adenylate cyclase still exhibited enhanced dissociation by native hormone, even though Scatchard analysis yielded a single, non-cooperative class of binding sites. This suggests that enhanced dissociation of bound hormone does not provide a demonstration of negatively-cooperative site-site interaction. Furthermore, nonlinear Scatchard plots, typical of TSH binding in normal thyroid, represent two classes of binding sites, of which the high affinity type is responsible for stimulation of adenylate cyclase.

Subunit composition, organic anion binding, catalytic and immunological properties of ligandin from rat testis.

Rat testicular and liver cystols contain ligandin as determined immunologically, and have high glutathione-S-transferase activity. Unlike liver cytosol, testicular cytosol does not contain protein components that bind bilirubin or sulfobromophthalein with high affinity. To investigate these effects, ligandin was purified to homogeneity from rat testis. Whereas rat liver ligandin consists of equal amounts of two subunits with molecular weights of 22,000 (Ya) and 25,000 (Yb), more than 90% of testicular ligandin consists of Yb. Rat testicular ligandin is immunologically similar to liver ligandin, and has identical glutathione-S-transferase activity, but lacks the capacity for high affinity binding of bilirubin and sulfobromophthalein. The amino acid composition and other properties of testicular ligandin are similar to those of the Yb subunit of liver ligandin. Sulfobromophthalein and bilirubin biphasically inhibit the glutathione-S-transferase activity of liver ligandin: initial high affinity inhibition is followed by reduced inhibition. Testicular ligandin has only low affinity inhibition kinetics. These results suggest that Ya is required for high affinity binding, and that reduced organic anion binding by testicular ligandin results from the lower amounts of Ya in testis.

Specific estradiol-17β binding component in adult rat kidney

An estradiol-17β (E 2) binding component has been identified in adult rat kidney which exhibits characteristics of a specific receptor. Scatchard analysis of kidney cytosol revealed a single class of binding sites for E 2, having high affinity (~0.6 × 10 10 M) and limited binding capacity (12–20 fmol/mg protein). The eytosol macromolecule has a sedimentation coefficient of approximately 4S in either low or high salt (0.4 M KCl) sucrose density gradients. Competition studies using a 100-fold excess of various unlabeled steroids demonstrated greatest inhibition by various estrogens, with E 2 and DES as the most effective competitors; whereas several androgens, aldosterone and progesterone inhibited binding very little. Incubation of kidney slices with [ 3H]-E 2 resulted in localization of the steroid in purified nuclei. Addition of a 100-fold excess of unlabeled E 2 or DES inhibited translocation of [ 3H]-E 2 by 94 and 79% respectively, whereas testosterone, dihydrotestosterone and aldosterone exhibited only minimal inhibition on nuclear translocation of [ 3H]-E 2 receptor. These data offer strong evidence for the existence of an E 2 receptor in rat kidney. It remains to be determined which specific cellular events are regulated by E 2 in the rat kidney.