High Affinity-low Capacity Sites Research Articles

High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6pmol/106cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350pmol/106cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

1,25-Dihydroxyvitamin D3 receptors and human colon adenocarcinoma

Epidemiological evidence suggests that dietary calcium and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) might have a protective effect against colorectal cancers. Since the presence of receptors is required for steroid action, specific 1,25-(OH)2D3 receptors (RD3) were investigated in biopsies taken at different levels of the colon. The study involved 90 biopsies from patients operated on for colorectal adenocarcinoma. They were paired biopsies from adenocarcinoma tissue and adjacent normal mucosa. In addition, 26 normal intestinal mucosa biopsies from patients without cancer were examined. RD3 receptors were assayed in tissue extract by the dextran-coated charcoal technique and also characterized by sucrose density gradient sedimentation. Scatchard analysis showed a single class of specific high affinity-low capacity sites binding for 1,25-(OH)2D3. The incidence of RD3 was 86 per cent in normal mucosa (n = 77) and lower in carcinoma (n = 34), for which the incidence decreased significantly (P less than 0.001) from right colon (58 per cent) to left colon (37 per cent) and rectum (19 per cent). These data suggest that the normal colon is a potential target organ for 1,25-(OH)2D3 which might modulate calcium transport in the colon. Loss of receptivity to 1,25-(OH)2D3 is associated with malignant transformation.

3H]p-Aminoclonidine and [ 3H]idazoxan label different populations of imidazoline sites on rat kidney

In the presence of RS-15385-197 to preclude binding to α 2-adrenoceptors, [ 3H]p-aminoclonidine labelled a low affinity high capacity site, (K d = 127.6±19.7 nM, B max 978±172 fmol/mg proteins) whereas [ 3H]idazoxan labelled a high affinity low capacity site (K d = 1.66±0.28 nM, B max 45.3±11.4 fmol/mg protein). Clonidine and p-aminoclonidine showed moderate affinity for the site labelled by [ 3H]p-aminoclonidine, but low affinity for the site labelled by [ 3H]idazoxan, whereas idazoxan showed high affinity for [ 3H]idazoxan and low affinity for [ 3H]p-aminoclonidine binding. Naphazoline inhibited [ 3H]idazoxan in a biphasic manner suggesting that [ 3H]idazoxan may label an heterogeneous population of imidazoline sites. GTP inhibited [ 3H]idazoxan but not [ 3H]p-aminoclonidine binding. These results suggest that [ 3H]idazoxan labelled imidazoline I 2 binding sites, whereas [ 3H]p-aminoclonidine labelled a novel subtype which showed marked differences to the imidazoline I 1 binding site reported in bovine and human brainstem.

Modulation by dexamethasone of insulin binding and insulin receptor mRNA levels in U-937 human promonocytic cells

Treatment with 5 × 10 −6 M dexamethasone stimulated insulin binding in human promonocytic U-937 cells. When curvilinear Scatchard plots were examined according to the one-site model, only changes in affinity, but not in receptor numbers, were observed. However, when the two-site model was applied, an increase in both the affinity and the number of the high affinity-low capacity sites was observed, with maximum values at 15 h. By contrast, the low affinity-high capacity sites did not undergo significant alterations. Northern blot assays revealed two insulin receptor-related mRNAs of approximately 11 and 7 kb in size. Dexamethasone increased the levels of these RNAs, following similar kinetics to those of high affinity receptor expression. This suggests that the 11 and 7 kb species carry information for high affinity insulin receptor, and that in U-937 cells the expression of this receptor subclass is primarily regulated at the mRNA level.

Increased insulin receptor binding in erythrocytes from growth hormone-deficient children

Erythrocytes from growth hormone-deficient children (GHd-children) (n = 10) showed a statistically significant increase in insulin binding at low unlabeled insulin concentrations, together with a threefold decrease in apparent receptor affinity, as compared to control children (C) (n = 11). Scatchard analysis of the binding data using the two-site model revealed that both the receptor concentration R1 [GHd-children 0.10 +/- 0.01 ng/ml and C 0.03 +/- 0.002 ng/ml] and the dissociation constant KD1 [GHd-children (0.48 +/- 0.05) x 10(-9) M and C (0.19 +/- 0.01) x 10(-9) M] for high affinity-low capacity sites were significantly increased in erythrocytes from GHd-children, while neither receptor concentrations (R2) nor the dissociation constant (KD2) for low affinity-high capacity sites proved to be altered. These events were accompanied by a normal sensitivity to insulin as well as glucose tolerance in the GHd-group. The meaning of the increased insulin binding with normal insulin sensitivity in GH-deficiency is discussed.

Evidence of 1,25-dihydroxyvitamin D 3-receptors in human digestive mucosa and carcinoma tissue biopsies taken at different levels of the digestive tract, in 152 patients

Epidemiological and experimental data suggest that dietary calcium and 1,25-dihydroxy vitamin D 3 (1,25-(OH) 2D 3) are protective against colorectal cancers, while their activity on colon mucosa still remains unknown. Since the presence of receptors is required for steroid action, specific 1,25-(OH) 2D 3 receptors were investigated in biopsies taken at different levels of the digestive tract from the oesophagus to the rectum and in pancreas. The total study involved biopsies from 152 patients. In 82% of the cases they were paired biopsies in adenocarcinoma tissue and in adjacent normal mucosa (NM). There were 120 operated on for colorectal adenocarcinoma (HCRA). 1,25-(OH) 2D 3 receptor was assayed in tissue extract by the dextran-coated charcoal (DCC) technique and also characterised by sucrose density gradient centrifugation. Scatchard analyses showed a single class of specific high affinity-low capacity sites binding for 1,25-(OH) 2D 3 with a K d = 1.48 ± 0.8 · 10 −10 M ( n = 119). The sedimentation coefficient of the steroid receptor complex was approximately 3.2 S. The incidence of 1,25-(OH) 2D 3 receptors was significantly higher in NM (82.5%) than in HCRA (34.5%). In HCRA this incidence decreased from right colon (64.7%) to left colon (27.7%) and rectum (15%). All positive HCRA in left colon and rectum ( 16 76 ) were histologically well differentiated. The receptor content in NM and HCRA was in the same range: (median) 10–314 (58) and 13–175 (64) fmol/mg protein. These data suggest that 1,25-(OH) 2D 3 may modulate calcium transport in colon, as in the intestine. Also, loss of receptivity to 1,25-(OH) 2D 3 is observed as associated with malignant transformation of the human colorectal mucosa.

Plasma protein binding of ceftriaxone.

1. The plasma protein binding characteristics of ceftriaxone, a new cephalosporin antibiotic, were determined in human, baboon, rabbit, dog and rat plasma. 2. The protein binding of ceftriaxone was similar and concentration-dependent in human, baboon, rabbit and rat plasma, being highly bound (90-95%) at low concentrations (less than 100 micrograms/ml) but considerably less bound (approx. 60%) at high concentrations (greater than 400 micrograms/ml). Binding in dog plasma was also concentration-dependent but much lower (approx. 25%) at lower concentrations (30 micrograms/ml) and virtually unbound (2%) at high concentrations (1 mg/ml) over a similar concentration range. 3. Binding of ceftriaxone to human plasma involved two sites: a high affinity-low capacity (saturable) site and a low affinity-high capacity site. Binding to dog plasma apparently was at a single, high affinity-low capacity site. 4. The pharmacokinetics of ceftriaxone in an animal species with binding characteristics similar to man (baboon), appear to be non-linear when based on total drug concentration and linear when based on the free drug concentration. In the dog, pharmacokinetic parameters did not change appreciably if calculated from total or free drug concentrations, due to the low protein binding.

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.

Vasoactive intestinal peptide binding to specific receptors on rat parotid acinar cells induces amylase secretion accompanied by intracellular accumulation of cyclic adenosine 3'-5'-monophosphate.

Specific binding sites for vasoactive intestinal polypeptide (VIP) were characterized in dispersed rat parotid acini. The binding of [125I]VIP was rapid, saturable, reversible, and temperature dependent. Scatchard analysis indicated two functionally independent classes of receptor sites: 41,000 high affinity-low capacity sites per cell with a dissociation constant (Kd) of 6.4 nM and 420,000 low affinity-high capacity sites per cell with a Kd of 150 nM. A peptide with N-terminal histidine and C-terminal isoleucine and secretin, which are structurally related to VIP, inhibited the tracer binding 30 and 200 times less strongly, respectively, than VIP. Epinephrine and carbachol did not inhibit [125I]VIP binding to parotid acinar cells. VIP stimulated cAMP accumulation in parotid lobules and induced amylase secretion in a dose-dependent manner. A peptide with N-terminal histidine and C-terminal isoleucine and secretin were less potent than VIP regarding cAMP accumulation (1/12 and 1/80 of VIP, respectively) and amylase secretion (1/40 and 1/500 of VIP, respectively). Substance P did not stimulate cAMP accumulation but stimulated amylase secretion more strongly than VIP. These observations clearly demonstrated the presence of VIP receptors coupled to adenylate cyclase system in the rat parotid gland, which plays an important role in the regulation of the amylase secretion. The regulation of parotid function by VIP was independent of the adrenergic or muscarinic regulatory system and of the influence of substance P.

3H]Oxotremorine-M binding to membranes prepared from rat brain and heart: Evidence for subtypes of muscarinic receptors

[ 3H]Oxotremorine-M has been used as a ligand to label muscarinic binding sites on membranes prepared from rat fore-brain and heart. In rat brain membranes, two binding sites could be identified: a high affinity low capacity site and a low affinity high capacity site. In contrast, only a high affinity site could be labelled in heart membranes. The potency order of agonists for the high affinity site in brain membranes and heart membranes was the same, suggesting that these binding sites represent the same subtype of muscarinic receptors. However, the affinity of pirenzepine for the brain high affinity site was higher than that for the heart high affinity site suggesting that these sites may represent different receptor populations. The potency order of agonists for the high affinity and low affinity sites in brain membranes were significantly different suggesting that these binding sites represent pharmacologically distinct binding sites. GTP abolished the high affinity sites in heart and brain membranes, but the low affinity site in brain membranes appeared unaffected. These results are consistent with the hypothesis proposing subtypes of muscarinic receptors.

Binding of noradrenaline to bovine serum albumin

The binding of noradrenaline to bovine serum albumin (40 mg ml −1 ) has been studied by gel filtration. Over the range of 5 × 10 −9 M to 10 −3 M of noradrenaline, the data obtained fit a model with three classes of noninteracting binding sites: a high affinity-low capacity site with a K A of 1.18 × 10 6 M −1 and a capacity of 11.84 pmol per mg of protein; an intermediate site with a K A of 7.42 × 10 4 M −1 and a capacity of 57.25 pmol mg −1 ; and a low affinity-high capacity site with a K A of 8.64 × 10 2 M −1 and a capacity of 1265 pmol mg −1 . There are no significant differences in binding among the three bovine serum albumin preparations considered. Both (±) and (−) noradrenaline are bound equally well by albumin. The binding is a relatively slow process but has a very rapid initial rate, about 40-fold higher than the observed at the half-maximal binding. At pH 7.4 the binding capacity is about 1.7-times higher and the initial rate is about 10-fold higher than at pH 5, but the affinity is similar. There is no degradation of noradrenaline in the presence of serum albumin. The reversibility of binding is small by dialysis or acidic precipitation. At physiological concentrations of noradrenaline and serum albumin it is estimated that about 60% of noradrenaline would be bound. The possible physiological significance of binding is discussed.

Demonstration of specific receptors for calcitonin in isolated trout gill cells

1. 1. Salmon calcitonin binding by isolated gill cells from rainbow trout, Salmo gairdneri has been investigated. 2. 2. The calcitonin receptor interaction is time- and temperature-dependent. 3. 3. 50% of inhibition of the 125I labeled calcitonin binding is observed in presence of 1.5 ng/ml unlabeled salmon calcitonin. 4. 4. Two types of receptors are described: a high affinity-low capacity site and a low affinity-large capacity site. 5. 5. These studies strongly support the role of calcitonin as a hormone regulating the gill function in physiological conditions.

Relationship between the (--)-[3H]-dihydroalprenolol binding to beta-adrenoceptors and transmembrane 86Rb efflux of the BC3H1 nonfusing muscle cell line.

1 We have studied the binding properties of the beta-adrenoceptor antagonist [3H]-dihydroalprenolol ([3H]-DHA) on a membrane preparation of the non-fusing muscle cells BC3H1. 2 [3H]-DHA appears to bind to two classes of sites. The first site has a high affinity (KD = 0.53 nM) and a low capcity (Bmax = 58 fmol/mg of protein). The second site has a low affinity (KD = 110 nM) and a high capacity (Bmax = 1100 fmol/mg of protein). 3 The pharmacological properties of the high affinity low capacity site correspond to the known properties of the beta 2-adrenoceptors since the agonists inhibit [3H]-DHA binding following the series isoprenaline greater than adrenaline greater than noradrenaline greater than phenylephrine and the antagonists following the series alprenolol congruent to propranolol greater than butoxamine greater than practolol greater than phentolamine. 4 The binding properties of the beta-adrenoceptors were correlated with the effect of beta-adrenoceptor agonists and antagonists on 86Rb efflux rate from BC3H1 Cells. 5 There is very good correlation between the dissociation constants obtained by inhibition of [3H]-DHA binding by the antagonists alprenolol and propranolol, and the inhibition constants calculated from their antagonism of the 86Rb efflux rate stimulation by adrenaline. The ratio of the dissociation constants obtained by inhibition of [3H]-DHA binding by agonists and their EC50, calculated from 86Rb efflux curves, is higher than 1. This high KD/EC50 ratio indicates a high coupling efficiency between receptor occupancy by agonists and the biological effect measured.

Binding of rat and bovine thyrotropin to specific sites in rat thyroid tissue.

Both 125I-labeled bovine TSH (bTSH) and rat TSH (rTSH) bind rapidly and saturably to a articulate fraction of thysroid glands of rats. Unlabeled bTSH and rTSH displaced 125I-labeled rTSH from binding sites in the thyroid particulate in an analogous manner. Specific binding of tracer concentrations of [125TJbTSH to the rat thyroid particulate fraction was several times higher than that of [125I]rTSH. Rat GH failed to displace 125I-labeled bTSH, rat FSH had only a slight inhibitory effect at the concentrations used, but rat LH displaced labeled bTSH better than bTSH did. Specific binding of [125I]bTSH to thyroid (21.6%) was several times greater than that to spleen (5.6%), liver (3.6%), or kidney (2.4%). At 37 C, maximum binding was achieved within 30 min but quickly decreased to lower levels. Incubation at temperatures above 4 C for 3 h resulted in a reduction in binding. At 4 C, binding oi [125I]bTSH to the particulate fraction w s rapid, saturable, and remained stable for 4.5 h but decreased within 21 h. Optimum binding occurred at pH 6.0, but experiments were conducted at pH 7.45. An excellent correlation coefficient (0.994) was apparent between maximum binding capacity, as determined from analyses of Scatchard plots, and particulate protein concentration. Scatchard analysis of bTSH binding was usually most consistent with the presence of a single binding site, although in some experiments a lower affinity, higher capacity site also seemed to be present. The mean association constant of the single binding site when one was found or the high affinity low capacity site when two sites were evident was 2.2 ± 0.5 10* M (mean ±SD) in four experiments. In the same experiments, the maximum binding capacity averaged 117.5 ± 43.0 juUμg, 2.9 ± 1.1 ng/μg, or 6.3± 2.3 × 10 ′ sitesμi.g particulate protein. The system here described, which makes use of a particulate fraction from the rat thyroid, may prove a valuable model for studying the role of alterations in TSH receptors in the response of the thyroid to a variety of stimuli

Effect of calcium on [125I]insulin binding to rat adipocytes.

The specific binding of [125I]insulin to rat adipocytes was reduced in the absence of extracellular calcium. The addition of calcium to the calcium-free medium during incubation restored [125I]insulin binding towards normal. The specific binding of insulin was significantly increased with calcium concentrations as low as 0.5 mM and maximal binding occurred with 5 mM calcium. Scatchard analysis of the data suggests two major binding sites, one a high-affinity low-capacity site (Kd, 1.5 X 10(10) M-1) and the other a lower-affinity high-capacity site (Kd, 4.7 X 10(9) M-1). There was a 50% decrease in the number of high-affinity sites in absence of extracellular calcium. The dissociation curve of receptor-bound insulin was nonlinear both in the absence and presence of extracellular calcium suggesting receptor heterogeneity. The dissociation rate of receptor-bound insulin was greater when insulin was bound in the absence of extracellular calcium than in its presence. These results indicate that extracellular calcium, by increasing the number of high-affinity receptor sites, can alter the the ratio of high-affinity to low-affinity receptors for insulin in adipocytes.

Quantitative analysis of tubulin and microtubule compartments in isolated rat hepatocytes.

A combined morphometric and biochemical approach has been used to identify and quantitate microtubules and tubulin in isolated hepatocytes. The total soluble pool of microtubule protein was estimated by specific high affinity binding to radiolabeled colchicine. Scatchard analysis of the data identified two populations of binding sites: high affinity-low capacity sites resembling tubulin and low affinity-high capacity sites believed to represent nonspecific colchicine-binding sites. Data from these studies indicate that tubulin represents 1% of the soluble protein of the cell, that 9.0 X 10(-14) dimers of tubulin are present per microgram soluble hepatocyte protein, and that the average hepatocyte contains 3.1 X 10(7) tubulin dimers. Our calculations suggest that this amount of tubulin would form a microtubule 1.9 cm in length if totally assembled. However, stereological measurements indicate that the actual length of microtubules in the cytosolic compartment of the average hepatocyte is only 0.28 cm. Thus, these experiments suggest that only 15% of the available tubulin in hepatocytes of postabsorptive rats is assembled in the form of microtubules.

Quantitative Aspects of the Insulin-Receptor Interaction in Liver Plasma Membranes

Abstract The interaction of insulin with a purified and well characterized preparation of liver plasma membranes has been studied. The time course of binding of 125I-insulin and displacement of tracer at the plateau of binding was measured as a function of temperature and ionic strength. Hormone tracer concentrations were in the low physiologic range. The reaction is complex and includes reversible binding of insulin to its receptor and degradation of both insulin and receptor. The degrading reactions were minimized by using low to medium membrane concentrations and were corrected for by measuring the intact insulin remaining. When the binding reaction was studied over the range of 0.06 to 1000 ng per ml of insulin at 30°, the data fit a model with a minimum of three classes of receptor sites: a high affinity low capacity site with a K of 2.0 x 109 m-1 and a capacity of 5 x 10-14 moles per mg of membrane protein; a low affinity-high capacity site with a K of 2.1 x 108 m-1 and a capacity of 1.5 x 10-12 moles per mg of membrane protein; and a very low affinity-high capacity site (nonspecific site) representing about 5% of the total tracer binding. Both the affinity and binding capacity are influenced by temperature and ionic strength. Kinetic data for insulin receptor complex dissociation are also consistent with heterogeneity of receptor sites. Discrepancies in the literature between the number of reported classes of insulin receptor sites and their affinity constants are discussed in terms of the variables of this complex reaction.