Direct assessment of beta-adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177. Evidence for agonist high-affinity binding complex and for beta 1 and beta 2 receptor subtypes

The characteristics of the binding of the hydrophilic beta-adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37 degrees C and 6 degrees C. At both temperatures and at 90% saturation, the non-specific binding was less than 30% of the total binding. At 37 degrees C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 +/- 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35 000 and 115 000 sites per cell in adipocytes with diameters of 60 microns and 88 microns, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective beta-antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for beta 1 and beta 2 adrenergic subtypes. In contrast, beta agonist competition curves, which modeled to two affinity-states of binding, showed high-affinity-state Kd values for agonists 10-25-times higher than those found in membranes under the same experimental conditions. At 6 degrees C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37 degrees C, the Kd value for (-)-isoproterenol binding to the high affinity state of these sites (3.0 +/- 0.5 nM) was 25-times lower than at 37 degrees C and similar to the value found in membrane preparations (1.5-4 nM). These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological beta-adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist-binding promotes a rapid and temperature-dependent conformational change of the beta-adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high-affinity complex.     

Two distinct affinity binding sites for IL-1 on human cell lines

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.     

Characterization and subcellular localization of GnRH analog binding in rat brain

The binding of a degradation-resistant analog of gonadotropin-releasing hormone, [D-Phe6]GnRH, to rat brain crude particulate preparation was studied. The binding of this analog at 0 degrees C was saturable and Scatchard analysis revealed the presence of 2 binding sites: one with KD = 1.39 x 10(-7) M and Bmax = 265 pmole/mg protein, and another of lower affinity but higher capacity with KD = 5.58 X 10(-6) M and Bmax = 1734 pmoles/mg protein. The binding at 0 degrees C was substantially higher than that obtained at 37 degrees C, due to binding site-inactivation processes occurring at 37 degrees C. The binding sites exhibited a considerable degree of specificity for GnRH as unrelated peptides (with the exception of ACTH) display a much weaker affinity than GnRH and GnRH analogs. Subcellular fractionation demonstrated that most of the binding was associated with the mitochondrial fraction.     

Multiple thyroid hormone binding sites on rat liver nuclear envelopes

Nuclear envelopes relatively free of plasma membrane contamination were isolated from the male rat liver. Equilibrium binding of T3 to nuclear envelopes occurred after incubation for 3 h at 20 degrees C. Scatchard analysis revealed two classes of binding sites; a high affinity site having a KD of 1.8 nM with a maximum binding capacity of 14.5 pmol/mg protein and a low affinity site having a KD of 152.1 nM with a maximum binding capacity of 346.8 pmol/mg protein. No degradation of the radioligand occurred during incubation with the nuclear envelope. T4, rT3 and Triac competed effectively for the binding of T3 to the high affinity site whereas only T4 competed well for binding to the lower affinity site. The binding site was protease sensitive but not salt extractable. Multiple T3 binding sites having similar affinities have been reported on plasma membranes. An intriguing possibility is that membrane binding sites may be involved in translocation of thyroid hormone across membrane barriers.     

Pathways in the binding and uptake of ferritin by hepatocytes

The binding and uptake of rat liver ferritin by primary cultures of rat liver hepatocytes was studied in order to assess the relative importance of saturable, high-affinity pathways and nonspecific processes in the incorporation of the protein by the cells. To minimize artifacts, ferritin not subjected to heat treatment and labeled in vivo with 59Fe was used. Binding to cell membranes was estimated from incubations performed at 4 degrees C. After 2 h, when a steady state in cell-associated ferritin had been achieved, approx. 4-10(4) binding sites per cell were observed, with an affinity constant for ferritin of 1 x 10(9) M-1. At 37 degrees C, the maximal uptake from these sites was 1.3 x 10(5) ferritin molecules/cell per h. For ferritin molecules bearing an average of 2400 iron atoms, this uptake amounts to 5 x 10(6) iron atoms/cell per min. Half-maximal uptake was achieved at a ferritin concentration, or KM1, of 3 x 10(-9) M. Although uptake rates at least a thousand times greater could be achieved by binding to the much larger number of low-affinity sites, the apparent KM2 for such 'nonspecific' uptake was 4 x 10(-7) M. At ferritin concentrations up to 2 nM, at least 90% of ferritin bound and taken up by hepatocytes involves saturable, high-affinity sites, presumably true ferritin receptors.     

Cyclo (His-Pro), a metabolite of thyrotropin-releasing hormone: specific binding to rat liver membranes

[3H]cyclo(His-Pro) bound with high affinity (59 nM) to a single class of sites in rat liver plasma membranes, without significant tracer degradation during equilibration for 60 min at 0 degrees C. Binding was specific and saturable (3.9 pmol/mg protein), and were increased by the addition of K+, Mg++ and Na+ at optimal concentrations, but not of Ca++ at all concentrations tested. In vivo administration of cyclo(His-Pro), but not thyrotropin-releasing hormone, to rats caused the downregulation of cyclo(His-Pro)-binding sites with decreases in specific binding numbers but did not change binding affinity.     

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.     

Specific 3,3′,5′-Triiodothyronine (Reverse T3) Binding Sites on Rat Liver Plasma Membranes: Comparison with Thyroxine (T4) Binding Sites

Abstract

The binding characteristics of thyroxine (T4), triiodothyronine (T3), and reverse T3 (rT3) to rat liver plasma membranes (RLPM) were examined to explore the interactions of thyroid hormones with cell surface receptors. Scatchard analysis suggested that all three ligands bound to two classes of binding sites. The high affinity rT3 binding sites appeared to be distinct from the high affinity T4 sites, on the basis of differing optimum physicochemical conditions for binding, and analog displacement studies. The higher affinity constant for T4 was 1.7 ± 0.2 × 10⁹ M^-1 (mean ± SEM) and binding capacity was 3.1 ± 0.3 pmol mg ^-1 protein whereas for rT3 binding the Ka was 2.5 ± 0.4 × 10⁸ M^-1 and capacity was 6.2 ± 0.9 pmol mg ^-1. (¹²⁵ I) T3 bound with lower affinity and T3 tracer was readily displaced by T4. Moreover, comparatively higher concentrations of T3 were needed to displace either radiolabeled T4 or rT3, suggesting that T3 was binding to both the T4 and rT3 sites with lower affinity. Marker enzyme studies on RLPM, of varying purity prepared by different methods, showed a positive correlation between the activity of the plasma membrane enzyme magnesium-stimulated ATPase and high affinity rT3 and T4 binding. Column chromatography of the radioligands, after dissociation from membrane binding sites, confirmed that the integrity of the hormones was not altered during association or dissociation. Our results raise the possibility that the high affinity T4 and rT3 binding sites on RLPM may be hormone receptors mediating biological actions at the membrane level.     

Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.     

Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts

The binding and internalization of 125I-labeled alpha 2-macroglobulin (125I-alpha 2M) was studied in cultured fibroblasts. Two classes of binding sites were detected on cell surfaces. One class corresponds to the previously described, high affinity and low capacity sites. The other class of binding sites may mediate uptake of high physiological blood levels of 125I-alpha 2M and has not been described previously. At 0 degrees C, this lower affinity class saturates at approximately 1,000 micrograms/ml and has a capacity of approximately 600,000 sites/cell. The lower affinity class accounts for the vast majority of cellular receptors for alpha 2M. An assay employing pepsin at pH 4 was developed to distinguish between surface-bound and internalized 125I-alpha 2M. Cellular uptake of 125I-alpha 2M at 37 degrees C has a component which saturates between 200 and 1,000 micrograms/ml and the rate of internalization of this component was approximately 1,700,000 molecules/cell/h. One mM Ca2+ was required for cell uptake of 125I-alpha 2M at 37 degrees C. Ca2+ was also required for binding at 0 degrees C to both low and high affinity classes of binding sites for 125I-alpha 2M. The transglutaminase inhibitors bacitracin, monodansylcadaverine, and N-benzyloxycarboxyl-5-diazo-4-oxonorvaline paranitrophenyl ester all inhibited cellular internalization of 125I-alpha 2M at 37 degrees C. Each of these three compounds selectively reduced 125I-alpha 2M binding to the high affinity, low capacity component at 0 degrees C. Based on the current binding studies and previous studies using electron microscopy which showed that bacitracin and other transglutaminase inhibitors block clustering of alpha 2M-receptor complexes in coated pits, we suggest that the inhibitors block the accumulation of occupied lower affinity alpha 2M receptors in coated pits where they acquire a higher apparent affinity.     

A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.     

The interaction of vasoactive intestinal peptide (VIP) with isolated bovine thyroid plasma membranes

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase were studied in bovine thyroid plasma membranes. The binding depended on time, temperature and was saturable and specific. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 13 nM) and low capacity (6411 sites/pg), and a class with low affinity (Kd = 480 nm) and high capacity (105,300 sites/pg) at 15 degrees C. Secretin, glucagon, insulin and somatostatin did not displace the tracer from the membranes. VIP stimulated cyclic AMP production. Maximal cyclic AMP production (2-fold above basal values) was observed with 100 nM VIP and half-maximal response was obtained at 5 nM VIP at 15 degrees C.     

Binding of oxytocin to uterine cells in vitro. Occurrence of several binding site populations and reidentification of oxytocin receptors

Myometrial and endometrial cells of sheep, rat, and calf in monolayer cell culture display at least three populations of binding sites for oxytocin, with dissociation constants (Kd) of approximately 5 X 10(-9), 4 X 10(-7), and greater than 10(-5) mol/liter, respectively. Binding of the tritium-labeled oxytocin (concentration range, 10(-11) to 5 X 10(-4) M) to the first two sites is displaceable by cold oxytocin. The ratio of binding capacities of the high to medium affinity site appears to average 1:18. Dissociation rate constants for these sites (22 degrees C) are roughly 10(-4) and 2 X 10(-3) s-1, respectively. The capacity of the low affinity site varies in individual cell preparations and is between 5 and 66 times that of the medium affinity site. The low affinity binding sites may not be fully saturable and may follow a nonasymptotic binding isotherm. Logarithms of Kd and binding capacity for individual binding sites are linearly correlated. The coexistence of the three sites was also proven by cluster analysis based on similarities between Kd, binding capacity, and Hill coefficient. Only minor systematic species and cell type differences occur in these properties. The value of Kd for the oxytocin receptor in rat myometrium, derived recently from a stepwise irreversible inhibition of uterotonic response to oxytocin, is close to 2.5 X 10(-7) mol/liter. Additional pharmacological data (pA2 values of structural analogues of oxytocin acting as competitive inhibitors) also reveal a Kd value of 3 X 10(-7). It is, therefore, concluded that the receptors for oxytocin in rat myometrium are identical with the medium affinity site.     

Characterization of high density lipoprotein binding activity in rat adrenocortical cells

Rat adrenocortical cells take up high density lipoprotein cholesterol for use as steroidogenic substrate. To better understand this unique uptake process, we have first characterized HDL binding. Infusion of human 125I-labeled HDL into rats pretreated with 4-APP demonstrated that the adrenal and ovary accumulate HDL in a saturable fashion in vivo. Subsequent studies using isolated rat adrenocortical cells demonstrated that cellular uptake of HDL is comprised of two events. One event is characterized by reversible membrane binding and is complete by 60 min (t1/2 = 20 min). The second event is marked by irreversible apoprotein accumulation which continues for at least 3 hr. Reversibly bound material exhibits the same apoprotein distribution as unincubated HDL. Irreversible accumulation could not be attributed to internalization or lysosomal accumulation inasmuch as it also occurred with partially purified plasma membranes and was not enhanced by addition of chloroquine. Reversible binding of human HDL3 exhibited a saturable dependence on concentration (Kd = 27 micrograms protein/ml; N = 3.0 X 10(6) sites/cell) similar to that previously reported for rat liver, ovary, and testis. Cell accumulation of HDL decreased by over 80% at 4 degrees C compared to 37 degrees C, did not require calcium, and was not diminished by prior cell treatment with trypsin or pronase. These results indicate that rat adrenocortical cells possess plasma membrane recognition sites for HDL with different properties than those of the LDL receptor. Moreover, adrenal accumulation of HDL apoproteins does not lead to secondary lysosome formation.     

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.     

Properties of vasoactive intestinal peptide-receptor interaction in rat liver membranes

The properties of the specific receptors for vasoactive intestinal peptide (VIP) in rat liver plasma membranes have been studied by using 125I-VIP as a tracer. The binding of the peptide was a reversible, saturable and specific process, as well as time and temperature dependent. Peptide inactivation was also dependent on time and temperature and remained relatively low in the standard conditions used, as it happened in the inactivation of the binding sites. The binding data were compatible with the existence of two classes of VIP receptors: a high affinity (Kd = 4.2 x 10(-10) M) and low binding capacity (1.5 pmol VIP/mg protein) class and another one of low affinity (Kd = 1.7 x 10(-7) M) and high binding capacity (38.6 pmol VIP/mg protein). The specificity of the binding sites of VIP was established from the fact that binding of 125I-VIP was inhibited by native VIP and by 60-fold higher concentrations of secretin but not by the parent hormone glucagon, by insulin or somatostatin at concentrations as high as 10(-6) M.     

Platelet-derived growth factor. II. Specific binding to cultured cells

We have prepared radioiodinated purified platelet-derived growth factor (125I-PDGF) which retains full mitogenic activity. The binding of 125I-PDGF to Swiss 3T3 cells is saturable and highly competed by whole blood serum, purified unlabeled PDGF, and by material from each stage in the purification of PDGF from platelet-rich plasma. Other purified mitogens and substances tested do not compete. 125I-PDGF binding to fibroblasts, 3T3 cells, and arterial smooth muscle cells shows an apparent dissociation constant of 10(-11) M, comparable to the range in which PDGF is mitogenic. A clone of Swiss 3T3 cells obtained from a population selected repeatedly against mitogenic response to PDGF shows a greatly reduced mitogenic response to PDGF and binds only 5% as much 125I-PDGF/cell. The binding capacity of the different cell types tested ranges from 2,500 binding sites/cell on the poorly responding variant to 390,000 binding sites/cell on one strain of Swiss 3T3 cells. Cell types that do not respond to PDGF do not show specific high affinity binding of 125I-PDGF. At 4 degrees C, 125I-PDGF binding to monolayer cultures is relatively slow. Equilibrium binding of low concentrations of 125I-PDGF is not achieved during 7 h unless the binding medium is constantly mixed. 125I-PDGF binding at 4 degrees C shows a broad pH optimum between 6.3 and 8.0. Binding does not seem to require Ca2+ or Mg2+ but is reduced more than 6-fold if both monovalent and divalent salts are omitted. The initial rate of 125I-PDGF binding is greater at 37 degrees C than at 4 degrees C but cell-associated 125I begins to decline soon after reaching a peak value at 30-60 min. Coincident with this decline, trichloroacetic acid-soluble 125I appears in the medium and the binding capacity of the cells declines. These phenomena suggest that PDGF and its receptor may be internalized and degraded.     

Receptor-mediated uptake and internalization of transthyretin

Evidence of cellular transthyretin (TTR) binding was sought because of the observation that transthyretin can increase the uptake of its hormonal ligand. Transthyretin was bound by human hepatoma (Hep G2) cells in a time- and temperature-dependent manner, reaching equilibrium within 2 h. Scatchard analysis was consistent with a single class of high affinity binding sites with a Kd of approximately 5 nM at 0 and 4 degrees C and 14 nM at 37 degrees C. These dissociation constants are more than 2 orders of magnitude lower than the concentration of transthyretin in human serum. The apparent capacity at 0 degrees C, corrected for internalized TTR, was approximately 20,000 sites/cell. Saturable, high affinity binding of human transthyretin was also demonstrable with rat primary hepatocytes and human renal adenocarcinoma, neuroblastoma, and transformed lung cells. Rat and human transthyretin were equipotent in displacing isotopically labeled, species-specific transthyretin from human hepatoma cells and rat primary hepatocytes, a finding that is consistent with the strong homology between rat and human transthyretin. Eighty-eight percent of the saturable uptake was internalized as determined by proteolytic removal of surface transthyretin. Internalization was dependent on receptor binding and was more markedly inhibited than surface binding at 0 degrees C. Concentrations of thyroxine within a range that saturated a significant proportion of the primary and secondary TTR iodothyronine binding sites increased the uptake and internalization of transthyretin in a dose-dependent manner. By analogy to the function of receptors for other transport proteins, the interaction between transthyretin and its receptor is likely to affect ligand delivery and may have additional metabolic effects.     

Specific binding of [3H]heparin to human carcinoma SW-13 and other mammalian cells

This study reports on the specific binding of [3H]heparin to human adrenocortical carcinoma cell line SW-13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between 90 and 120 min at 22 degrees C. Scatchard analysis revealed two classes of binding sites. The apparent Kd for high-affinity receptors is 2.14 x 10(-8) M with 1.48 x 10(6) sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin.