Studies of corticotropin receptors on rat adipocytes

Synthetic [¹²⁵I]-Tyr²³, Phe², Nle⁴-adrenocorticotropin (ACTH)-(1–38) ([¹²⁵I]-ACTH analog) with full biological potency and near theoretical specific radioactivity (1800 ± 75 Ci/mmol) was used to investigate ACTH receptors on isolated rat adipocytes derived from 42-day-old rats. Binding to adipocytes was studied in the presence of 1% bovine serum albumin (BSA) as well as 4% BSA. The interaction of the [¹²⁵I]-ACTH analog with adipocytes was highly specific, rapid, saturable, and reversible. Scatchard analysis of the binding data obtained in medium containing 1% BSA revealed a single class of binding sites with an apparent K_D = 170 ± 11.9 pM. Competition experiments with unlabeled ACTH also yielded a comparable value for the apparent K_D (143 ± 16.5 pm). The number of receptors per adipocyte was quite low (521–841/cell). The stimulation of lipolysis by ACTH was closely correlated with the binding, the apparent K_m being 145–177 pm. At a concentration of 4% BSA in the incubation medium, the binding curve was shifted significantly to the right (apparent K_D = 446 ± 77 pM) and the binding capacity was also significantly enhanced (1663 ± 208/cell) without any change in the apparent K_m for glycerol release (187 ± 7.1 pm).     

Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-³H₂]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 ± 0.008 min ¹ using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 ± 0.00013 min ¹. An association rate constant of 7.3 × 10⁶ M ¹ min ¹ was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B_max = 23.87 ± 1.15 pmol/mg protein, K_d = 18.1 ± 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC₃₀ in parentheses): azadirachtin (1.55 × 10⁻⁸ M) > dihydroazadirachtin (3.16 × 10⁻⁸ M) > dansyl dihydroazadirachtin (7.40 × 10⁻⁸ M) > DNP-azadirachtin (7.50 × 10⁻⁸ M) > biotin dihydroazadirachtin (1.27 × 10⁻⁷ M) ≫ 11-methoxy 22,23-dihydroazadirachtin (6.67 × 10⁻⁷ M). Arch. Insect Biochem. Physiol. 34:461–473, 1997. © 1997 Wiley-Liss, Inc.     

Cytochalasin B binding by human platelets

Intact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 10⁴ molecules/cell and dissociation constants (K_D) ranging from 2 × 10^?8 to 10^?6 M. Up to 85% of this saturable binding is displaced by 10^?5 M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with K_D similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with K_D ≌ 4 × 10^?7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with K_D ranging from 2.4 × 10^?8 to 1.5 × 10^?6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D-glucose and has a K_D of 1.5 × 10^?6M, while only 10–15% is CE-sensitive with a K_D of 2.4 ± 10^?8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Androgen receptor complex binding in murine skeletal muscle nuclei

Examination of binding of androgen-receptor complexes from murine skeletal muscle cytosol was performed by modified nuclear retention assay and modified nuclear acceptor assay. The experiments showed the binding of androgen-receptor complexes to the nuclear acceptor sites to be a cooperative process. Hill analysis of the data obtained resulted in a Hill coefficient of 3,6. The apparent dissociation constant for binding of cytosolic [³H]-testosterone-receptor complexes to nuclei was found to be in the range of K_D = 6 ? 8 × 10^?11 M. The nuclear matrix was able to bind androgen-receptor complexes in a saturable way, too.     

Histamine H-1 receptors on rat peritoneal mast cells

In order to characterize the receptor subtype involved in histamine stimulation of increased cyclic AMP levels in rat mast cells with consequent impairment of anaphylactically induced mediator release, the binding of the H-1 receptor antagonist [³H] pyrilamine to mast cells was examined. Pyrilamine bound rapidly, in a saturable and reversible fashion, and with increased binding at 4°C as compared with 21°C and 37°C. [³H] Pyrilamine binding was displaced by H-1 antagonists (tripelnnamine > yrilamine ≧ iphenhydramine) > histamine > the H-2 antagonist, cimetidine. H-1 agonists displaced pyrilamine binding less efficiently than histamine but better than H-2 agonists. Rat mast cells have a single homogeneous population of low affinity (K_D = 222 ± 33 nM) H-1 receptors with a Bmax of 9.7 ± 2.3 pm/10⁶ mast cells and 5.4 ± 0.92 × 10⁶ binding sites per mast cell. Thus, the mast cell has an H-1 type histamine receptor which is probably involved in histamine-induced cyclic AMP increases.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ 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 β₁-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.     

Stereoselective binding of isradipine to human plasma proteins

Isradipine (PN 200–110) is a highly potent calcium entry blocker with an asymmetrically substituted dihydropyridine ring (methyl- and isopropylester, respectively). The binding of the (+)-(S)-isradipine and (?)-(R)-isradipine to isolated human serum albumin (HSA, 30 μmol/l) and α₁-acid glycoprotein (AAG, 10 μmol/l) has been studied in vitro over a wide range of isradipine concentrations (0.06–20 μmol/l) using high-performance liquid chromatography (HPLC). HPLC experiments revealed that both isradipine enantiomers were bound to one class of high-affinity binding sites on the AAG molecule (n_(S) = 0.83 ± 0.05, K_a(S) = (1.33 ± 0.25) × 10⁶ 1/mol, n_(R) = 0.85 ± 0.07, K_a(R) = (1.17 ± 0.44) × 10⁷ l/mol). The (R)-enantiomer also exhibited an interaction with the secondary low-affinity binding sites (n′K′_{a (R)} = (2.66 ± 0.65) × 10⁴ l/mol). In contrast, the pharmacologically more potent (+)-(S)-enantiomer was more strongly bound to HSA than its optical antipode (n_(S) = 1.07 ± 0.07, K_a(S) = (1.76 ± 0.26) × 10⁵ l/mol, nK_a(R) = (3.62 ± 0.06) × 10⁴ l/mol). In general, the resulting binding characteristics of individual isradipine enantiomers showed stereoselectivity, but this was opposite for the two most important plasma binding proteins. The process of accumulation of isradipine by human platelets in the therapeutically relevant range (10–80 ng/ml) at 37°C was devoid of stereoselectivity. © 1995 Wiley-Liss, Inc.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Revised model of calcium and magnesium binding to the bacterial cell wall

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca²⁺, Region I has a K_A = (1.0 ± 0.2) × 10⁶ M^?1 and Region II has a K_A = (0.075 ± 0.058) × 10⁶ M^?1. For Mg²⁺, K_A1 = (1.5 ± 0.1) × 10⁶ and K_A2 = (0.17 ± 0.10) × 10⁶. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η₂, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca²⁺ and Mg²⁺ respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.     

Interaction of epidermal growth factor with adult rat liver parenchymal cells in primary culture

Adult rat liver parenchymal cells in primary culture exhibit specific saturable binding of ¹²⁵I-labeled murine epidermal growth factor (EGF). The Scatchard plot of the binding data obtained at 36 °C was curvilinear yielding two apparent dissociation constants of 1.5 × 10^?10m and 1.2 × 10^?9m with 27,000 and 57,000 sites per cell, respectively. The binding data obtained at 2 °C yielded a linear Scatchard plot with an apparent dissociation constant of 4.4 × 10^?9m and 78,000 sites per cell. Exposure of the hepatocytes to EGF at 36 °C resulted in a loss of EGF binding capacity due to down regulation of receptors. The cells recovered the capacity to bind EGF upon incubation in medium which did not contain EGF; this recovery was inhibited by cycloheximide. The cultures appeared to internalize and degrade bound EGF at 36 °C but not at 2 °C. The degradation of EGF was inhibited by chloroquine, an inhibitor of lysosomal enzymes. These data indicate that liver specifically binds and further processes EGF, and therefore, may be a physiological target tissue for this growth factor.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [³H]tyrosyl-PBAN28-33NH₂ to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO₃ ⁻ ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K_d of 5.73 ± 1.05 × 10⁻⁶ M and a Bmax of 1.85 ± 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH₂ and PBAN28-33ΝΗ₂ with a K_i of 4.3 ± 1.1 × 10⁻⁶ M and 4.9 ± 2.6 × 10⁻⁶ M, respectively. Accepted: 4 February 1999     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

Prostaglandin E2 receptor in the myometrium: distribution in subcellular fractions

[³H]Prostaglandin (PG) E₂ bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE₂ and PGE₁ competed for the [³H]PGE₂ binding site. The PGs inhibited in the following decreasing order: PGE₂ = PGE₁ ? PGF_2α > PGA₂ > PGF_1β > PGB₂. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 ± 0.14 × 10^?9. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10^?11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10^?11 mol binding sites/g. The results suggest that there is a high-affinity PGE₂ receptor present in both plasma membrane and sarcoplasmic reticulum.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

BINDING OF [99mTc]CHONDROITIN SULFATE TO SCAVENGER RECEPTORS ON HUMAN CHONDROCYTES AS COMPARED TO BINDING OF OXIDIZED [125I]LDL ON HUMAN MACROPHAGES

ABSTRACT

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with ^99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37°C for 2?h revealed the presence of high-affinity binding sites for CS with a K_d of 2.3 × 10^?9?mol/L and a B_max of 5.0 × 10⁸. Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 ± 19.5% and of the number of CS binding sites/cell by 62.0 ± 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [¹²⁵I]-LDL binding we found a K_d of 0.45 × 10^?8?mol/L and a B_max of 0.14 × 10⁶ on quiescent monocytes and for [¹²⁵I]-(ox)LDL binding a K_d of 1.8 × 10^?8?mol/L and a B_max of 1.3 × 10⁶ using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein–proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.