Concentration Of Specific Binding Sites Research Articles

Characterization of the 4 S polycyclic aromatic hydrocarbon-binding protein in human liver and cells

The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a soluble protein that binds PAHs with high affinity in mouse, rat, and rabbit. Until now, this protein had not been detected in human placenta or human cells in culture by cytosol labeling and gradient centrifugation assay. Thanks to a preliminary fractionation of cytosol by sedimentation on sucrose gradient or/ and gel permeation chromatography, we found that PBP was present in liver, MCF-7 cell line, and hepatocytes of human. To accurately quantitate PBP binding and determine specific binding parameters, a reduction in the amount of charcoal used to adsorb nonspecifically bound benzo[ a]pyrene was required. By saturation analysis, the concentration of specific binding sites for [ 3H]BP in PBP fraction from human liver was 4.6 pmol/mg of protein compared with 14.7 ± 1.4 pmol/mg in the same fraction from DBA/2J mouse liver. Kinetic studies analyzed by Scatchard and Woolf plots indicate that human liver and MCF-7 cells contain a low-affinity PBP form: the K d derived from Woolf plot analysis were 14.2 ± 1.4 and 26.2 ± 1.8 n m, respectively. DBA/2J mouse possesses a higher-affinity PBP form, the same analysis indicating a K d of 6.1 ± 0.3 n m. These data demonstrate that, by comparison to the mouse liver, a lower-affinity form of PBP is present in reduced concentration in human liver, explaining the impossibility of detecting this protein by sedimentation of human cytosol in sucrose gradient.

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 ± 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [ 3H]TCDD and 3-[ 3H]methylcholanthrene. 3-[ 3H]Methylcholanthrene did not bind to any component besides that at ∼9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [ 3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [ 3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant ( K d ) for binding of [ 3H]TCDD to Hep G2 Ah receptor was 9 n m by Woolf plot analysis, about an order of magnitude weaker than the affinity of [ 3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [ 3H]TCDD·Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [ 3H]TCDD at 37 °C in culture, sedimented at ∼6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC 50 for AHH induction was 5.3 /gm m for benz( a)anthracene and 1.3 μ m for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 m m sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drugmetabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.

Demonstration of a high affinity receptor for 1,25-dihydroxyvitamin D 3 in rat pancreas

The existence of a high affinity receptor for 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) in rat pancreas was biochemically demonstrated in this study. In order to study the properties of this putative receptor, we took advantage of the analysis of low ionic strength chromatin-localized 1,25(OH) 2D 3 receptor. Using this method, the susceptibility of receptor protein to enzymatic degradation was so decreased, and the contamination by plasma vitamin D binding protein (DBP) component was so efficiently eliminated that a specific, saturable binding for 1,25(OH) 2D 3 could be demonstrated in the saturation analysis and the peak for the receptor was consistently apparent in the sucrose density gradient analysis. The equilibrium dissociation constant (Scatchard K d ) was found to be 3.7 ± 1.5 × 10 -10 (M), and the concentration of specific binding sites was calculated to be 1.22 ± 0.40 (fmol/mg protein). The number of specific binding sites in the rat pancreas was only 0.44% of that present in rat intestine (277 ± 19 (fmol/mg protein)) and 6.7% of that in rat kidney (18.1 ± 1.0 (fmol/mg)). However, when a correction is made for the 1,25(OH) 2D 3 receptor distribution in the tissues and expressed as the receptor concentration per receptor-containing cells, the rat pancreatic receptor level was calculated to be about 30% of the rat intestine. Sucrose density gradient sedimentation of this receptor yielded a value of 3.2 ± 0.1 (S) for the sedimentation coefficient and this peak was displaceable by a 100-fold excess of nonradioactive 1,25(OH) 2D 3. These data provide evidence for the presence of a specific 1,25(OH) 2D 3 receptor in mammalian pancreas, and we suggest, in conjunction with the facts that vitamin D 3 and its active metabolites play a physiological role on insulin secretion from rat pancreatic β-cells, that this receptor might be involved in the mechanisms of action of vitamin D 3 on insulin secretion from rat pancreas via a genomic effect.

Localization of binding sites for insulin-like growth factor-I (IGF-I) in the rat brain by quantitative autoradiography

In vitro quantitative autoradiography was used to localize IGF-I binding sites in rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.01 nM 125I[Thr 59]IGF-I, alone or mixed with 10 nM unlabeled [Thr 59]IGF-I or insulin, for 22 h at 4 °C and apposed to LKB Ultrofilm. Measurement of labeled [Thr 59]IGF-I binding by computer digital image analysis of the autoradiographic images indicated that high affinity IGF-I binding sites are widely distributed at discrete anatomical regions of the brain microarchitecture. The highest concentration of specific binding sites was in the choroid plexus of the lateral and third ventricles. Unlabeled porcine insulin was less potent than unlabeled IGF-I in competing for binding sites on brain slices. Regions of the olfactory, visual, and auditory, as well visceral and somatic sensory systems were labeled, in particular the glomerular layer of the olfactory bulb, the anterior olfactory nucleus, accessory olfactory bulb, primary olfactory cortex, lateral-dorsal geniculate, superior colliculus, medial geniculate, and the spinal trigeminal nucleus. High concentrations of IGF-I-specific binding sites were present throughout the thalamus and the hippocampus, (dentate gyrus, Ca1, Ca2, Ca3). The hypothalamus had moderate binding in the paraventricular, supraoptic, and suprachiasmatic nucleus. Highest binding in the hypothalamus was in the median eminence. The arcuate nucleus showed very low specific binding, approaching the levels found in optic chiasm and white matter regions. Layers II and VI of the cerebral cortex also had moderate IGF-I binding. The results suggest that the development and functions of brain sensory and neuroendocrine pathways may be regulated by IGF-I.

Changes in vascular vasopressin receptors and responsiveness in DOCA/NaCl-treated rats.

This study investigated the effects of treatment with desoxycorticosterone acetate and salt (DOCA/NaCl) on blood pressure, aortic vascular vasopressin receptors and in vitro vascular responsiveness in male rats. Four groups of animals were utilized in the study: a control group on normal tap water, a control group drinking a 1% NaCl solution; a DOCA/NaCl-treated group (1% NaCl in the drinking water); and a uninephrectomized (1K) DOCA/NaCl-treated group. DOCA/NaCl treatment for 4 weeks resulted in a significant elevation in blood pressure which was more pronounced in the uninephrectomized animal. The concentration of specific binding sites for vasopressin on the aorta were reduced in both the DOCA/NaCl-treated groups. However, the vascular responsiveness of the aorta to vasopressin was significantly reduced only in the hypertensive, uninephrectomized-DOCA/NaCl-(1K DOCA/NaCl) treated rat. Both the maximal contraction and the sensitivity was reduced in the 1K DOCA/NaCl group. The results of this study would suggest that the vascular alterations to vasopressin are probably post receptor mediated and result from the DOCA/NaCl-induced hypertension.

Quantification of naloxone binding sites in brains from suckled beef cows during postpartum anestrus and resumption of estrous cycles.

Thirty beef cows, approximately 3 yr of age, were randomly assigned to be slaughtered on d 7, 14, 28, 42 or 56 postpartum. Each cow suckled one calf until slaughter. Data from cows slaughtered on d 42 and 56 were pooled and further classified as anestrous or cyclic based on the presence of a corpus luteum and elevated serum concentrations of progesterone at slaughter. Specific binding of [3H]naloxone (3H-NAL) to homogenates of tissue from hypothalamus (HYP), preoptic area (POA) and basal forebrain (BF) was assessed using multiple-point Scatchard analyses. Nonspecific binding was estimated in the presence of 10(-6) M naloxone. Separation of bound from free 3H-NAL was achieved by centrifugation at 20,000 X g. Concentration (fmol/mg original tissue wet wt) of 3H-NAL binding sites in POA tissue was higher (P less than .05) on d 28 postpartum in anestrous cows than in cyclic cows on d 42 + 56 postpartum (2.58 +/- .32 vs 1.58 +/- .10). When all anestrous cows were compared with cyclic cows, concentrations of 3H-NAL binding sites in POA tissues and in BF tissue were higher (P less than .05) in anestrous cows (anestrous POA, 2.12 +/- .17, cyclic POA, 1.58 +/- .10; anestrous BF, 2.94 +/- .41, cyclic BF, 2.19 +/- .16). Compared across brain regions for all cows, the concentration of specific binding sites for 3H-NAL was greater (P less than .01) in BF (2.5 +/- .2) than in POA (1.9 +/- .1) and greater (P less than .01) in POA than in HYP (1.5 +/- .1).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of glucocorticoid receptors in the kidney of immature and mature male rats

1. 1. Specific binding of [ 3H]dexamethasone to cytosol and the activation of bound hormone-receptor complexes were studied in the kidney of immature (3-week) and mature (26-week) Long-Evans male rats. 2. 2. The concentration of specific binding sites was significantly higher (25%) in the kidney of immature rats as compared with mature, while dissociation constants ( K d) remain unaltered at both ages. 3. 3. Heat activation (25°C for 45 min) significantly enhanced the binding of [ 3H]dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both ages to the same extent. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice versa) gave similar results to the non-mixed groups. 4. 4. Ca 2+ activation (0°C for 45 min with 20 mM Ca 2+) also enhanced the nuclear and DNA-cellulose binding at both ages but to a greater magnitude in immature rats. 5. 5. Differences in the number of specific binding sites and some of the physicochemical properties of kidney glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.

Age-dependent regulation of glucocorticoid receptors in the liver of male rats

Specific binding of [ 3H]dexamethasone to cytosol and the activation of bound hormone-receptor complexes were studied in the liver of immature (3 weeks old) and mature (26 weeks old) Long-Evans male rats. The concentration of specific binding sites was significantly higher (33%) in the liver of immature rats as compared to mature, while dissociation constants ( K d) remain unaltered at both ages. Heat activation (for 45 min at 25° C) significantly enhances the binding of [ 3H]dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both the ages, with a greater magnitude in mature rats. Cross mixing experiments (i.e., binding of activated cytosol from mature rats to nuclei of immature and vice-versa) show receptor specificity. Ca 2+ activation (20 mM Ca 2+ for 45 min at 0° C) also enhances the nuclear and DNA-cellulose binding at both the ages, but to a similar extent. Differences in the number of specific binding sites and some of the physicochemical properties of glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.

Rabbit uterine oxytocin receptors and in vitro contractile response: abrupt changes at term and the role of eicosanoids.

We examined the relation between increased uterine oxytocin receptor concentration and increased in vivo sensitivity of the rabbit uterus to oxytocin at the end of gestation. We determined oxytocin receptor concentrations in myometrium and decidua on different days near term of gestation and postpartum. We also examined the in vitro contractile response to oxytocin on days 30 and 5 days postpartum, when the uterus is unresponsive in vivo, and on day 31 (term), when the uterus is exquisitely sensitive to this hormone in vivo. In addition, we tested the role of endogenous eicosanoids and decidual oxytocin receptors in the myometrial contractile response to oxytocin by examining the contractile response in the presence of the cyclooxygenase/lipoxygenase inhibitor sodium meclofenamate or in muscle strips from which the decidua had been removed by scraping. The concentration of specific binding sites for [3H]oxytocin in myometrial and also decidual membrane preparations was determined. We demonstrate that contractile sensitivity to oxytocin increases at least 4-fold between days 30 and 31 (term) of gestation, and this is accompanied by a nearly 10-fold increase in the concentration of oxytocin-binding sites in both decidua and myometrium. The lesser sensitivity to oxytocin on day 30 was, however, only apparent in the presence of meclofenamate, which suggests that endogenous eicosanoids contribute to the preterm response to oxytocin measured in vitro. The maximal response to oxytocin (integrated area) increased 2-fold between day 30 and term. Thus, an increase in both sensitivity and maximal response to oxytocin could be demonstrated at term in vitro. Five days after parturition, maximal response and uterine sensitivity measured in the presence of meclofenamate had returned to those of the preterm uterus, and the concentration of oxytocin-binding sites had declined. In contrast, sensitivity and maximal response to the cholinergic agonist carbamylcholine declined between day 30 and term. These results support a highly regulated physiological role for oxytocin in parturition which depends primarily on changes in receptor concentration.

Sex hormone receptors in the hypothalamus and their role in sexual differentiation of the male rat brain

In this investigation, changes in the level of receptors for sex hormones in the hypothalamus and cerebral cortex of male rats were studied on the first through fifth days of postnatal life, and the results obtained were compared with the levels of luteinizing hormone and sex hormones in the peripheral blood in order to discover any correlation between these parameters. 2,4,6,7,-/sup 3/H-estradiol-17..beta.. and 1,2,6,7-/sup 3/H-testosterone were used as labeled hormones. The values of the association constant and concentration of specific binding sites for estradiol and testosterone in hypothalamus and cerebral cortex of male rats during neonatal development is shown. It is found that in male rats on the first day after birth, receptors for estradiol and testosterone are present and they enable the action both of the testicular hormone and that of estradiol to be realized.

An investigation on the effect of oligomycin on state-4 respiration in isolated rat-liver mitochondria

The inhibitory action of oligomycin on State-4 respiration in rat-liver mitochondria has been investigated in detail with regard to the extent, mode and characteristics of the inhibition. The possibility that this effect may be due either to some damage of the mitochondrial preparation used or to the presence of heavy contaminations by microsomes has been excluded. It has been found that the concentration of specific binding sites is the same in State 4 as in State 3. The extent of the inhibition appears to be related to the ADP concentration, rather than to ATP ADP ratios. The inhibition of this antibiotic on State-4 respiration does not depend on the experimental conditions used (i.e., choice of substrates or composition of the reaction medium). In agreement with these observations, it has been found that the membrane potential of State 4 is significantly increased when oligomycin is added. All these results provide further evidence to the conclusion that a large portion of State-4 respiration is linked to phosphorylation.

Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport

The binding of radioactively labelled atrazin, metribuzin and phenmedipham by broken chloroplasts was studied. From the double-reciprocal plots (bound vs. free inhibitors) a high affinity binding reaction is graphically isolated which is related to the inhibition of photosynthetic electron transport. It is concluded that the specific binding sites correspond to the electron carrier molecules which are attacked by the inhibitors. The relative concentration of specific binding sites is 1 per 300–500 chlorophyll molecules. The binding of the labelled substances is competitively inhibited by each of the indicated unlabelled substances, by DCMU and by several pyridazinone derivatives. These results suggest that triazines, triazinones, pyridazinones, biscarbamates and phenylureas interfere with the same electron carrier of the photosynthetic electron transport chain, according to the same molecular mechanism.

Binding of HQNO to beef-heart sub-mitochondrial particles

1. The fluorescence spectra of HQNO (2- n-heptyl-4-hydroxyquinoline- N-oxide) in water at pH 7.5 show an emission maximum at 480 nm and an excitation maximum at 355 nm. 2. The fluorescence is enhanced by binding to bovine serum albumin, and is completely quenched by binding to sub-mitochondrial particles of beef heart. 3. Binding experiments reveal specific binding of HQNO to sub-mitochondrial particles with a dissociation constant of 64 nM and, depending on the protein concentration, a considerable amount of aspecific binding. 4. The concentration of specific binding sites for HQNO is identical with that of antimycin-binding sites. Furthermore, the presence of antimycin prevents the binding of HQNO and antimycin releases HQNO from its binding site. 5. The binding of HQNO is not sensitive to the redox state of the respiratorychain components. 6. Inhibition of electron transfer by HQNO is caused by binding to the specific binding site. 7. The relation between inhibition of NADH or succinate oxidation and saturation of the binding site is hyperbolic. 8. The increase in the reduction level of cytochrome b on addition of HQNO in the presence of succinate and oxygen, either in the presence or absence of cyanide, does not parallel the inhibition of overall electron transfer. 9. All data can be quantitatively described and analysed using the model for electron transfer proposed by Wikström and Berden in 1972 (Wikström, M. K. F. and Berden, J. A. (1972) Biochim. Biophys. Acta 283, 403–420).

Glucocorticoid Receptors in Fetal and Adult Rabbit Tissues

Abstract The specific interaction of glucocorticoids with fetal and adult rabbit tissues was studied in vitro. Specific cytoplasmic binding sites for [3H]dexamethasone were demonstrated in liver, thymus, kidney, lung, muscle, heart, small intestine, skin, brain, and placenta of the rabbit fetus as well as in liver, thymus, spleen, kidney, small intestine, and lung of the adult rabbit. In the rabbit fetus at 28 to 30 days of gestation the concentration of specific binding sites for [3H]dexamethasone was higher (1.62 to 1.83 pmoles per mg of DNA) in placenta, kidney, and lung, somewhat lower (0.69 to 1.25 pmoles per mg of DNA) in skin, muscle, small intestine, and heart, and much lower (0.35 to 0.49 pmole per mg of DNA) in liver, thymus, and brain. Sucrose density gradient analysis of tissue extracts showed the presence of one or two [3H]dexamethasone-macromolecular complexes sedimenting at 7 to 8 S and 4 S. The 7 to 8 S complex was present in all tissues, but the 4 S complex was detected only in liver, kidney, small intestine, and spleen. These results demonstrate the presence of similar [3H]-dexamethasone-binding components (receptors) in a variety of fetal and adult rabbit tissues. It is suggested that in addition to the liver and thymus, which are usually regarded as the target tissues for glucocorticoids, several other rabbit tissues may also be targets for these hormones.

Demonstration and partial characterization of a rabbit serum protein which binds testosterone and dihydrotestosterone.

A protein in the serum of the adult male rabbit which binds testosterone and dihydrotestosterone is demonstrated. It is salted out by 50% ammonium sulfate and has a greater Rf than the analogous human protein at pH 9.5 in polyacrylarride gel electrophoresis. It can be separated from rabbit corticosteroid-binding globulin on Sephadex G200 and polyacrylamide gel electrophoresis. The concentration of specific binding sites in pooled serum is 1.8 X 10-7M and the association constant for dihydrotestosterone at 4C is 1.4 X l-9M-1. A number of steroids were examined for their ability to compete with testosterone for binding sites. In contrast to the human protein, estradiol was an extremely poor competitor. (Endocrinology 92: 1700, 1973)