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.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

ADSORPTION AND UPTAKE OF COPPER BY THE GREEN ALGA SCENEDESMUS SUBSPICATUS (CHLOROPHYTA)1

Copper (II) accumulation has been investigated in the green alga Scenedesmus subspicatus G. Brinkmann considering both adsorption and uptake kinetics. Experiments were conducted in a Cu- and PH-buffered medium at different free Cu²⁺ concentrations that were neither growth limiting nor toxic. We distinguished between adsorption on the cell surface and intracellular uptake by extracting copper from the cells with EDTA. Data from short-term experiments were compared with data obtained from experiments under steady state conditions. The accumulation of Cu can be described by two processes, an initial fast adsorption occurring within a minute followed by a slower intracellular uptake. Metal uptake followed Michaelis-Menten kinetics and is mediated by two systems, one with low and the other with high affinity. The maximum uptake rates (1.30 × 10^?-¹⁰ mol·[g dry wt algae]^?1· min^?1, 3.67 × 10^?-⁹ mol·[g dry wt algae]^?1·min^?1), and the half-saturation constants (6.84 × 10^?-¹⁴ M, 2.82 × 10^?-¹² M) for the two uptake systems were determined using the Lineweaver-Burk plot. The calculated maximum concentration of binding sites on the surface of the algae is initially higher (9.0 × 10^?-⁶ mol Cu.[g dry wt algae]^?1) than under steady state conditions (2.9 × 10^?-⁶ mol Cu·[g dry wt algae]^?1). This suggests that the initial binding to the algal surface comprises the binding to specific transport ligands as well as to inert adsorption sites. The conditional stability constant of the Cu binding to surface ligands was calculated as log K_Cu= 11.0 at pH 7.9. This freshwater alga has a high ability to accumulate Cu, reflecting its adaptation to the bioavailable concentration of copper.     

ALTERATIONS IN THE BINDING OF [3H]LEUCINE-ENKEPHALIN TO STRIATAL MEMBRANES BY ENDOGENOUS FACTORS

—Saturation binding studies with [³H]leu-enk ([tyrosyl-3, 5-³H(N)]⁵leu-enkephalin) revealed the presence of high and low affinity binding sites in a paniculate fraction derived from rat striatum. The binding of [³H]leu-enk to the high affinity component (K_D= 2.0 ± 0.3 nM) was sensitive to morphine and levorphanol, while the binding to the low affinity component (K_D= 21 ± 2 nM) was not. Incubation of the membranes, prior to assay for 30 min at 37°C, followed by centrifugation at 27, 000 g for 20 min in order to pellet the membranes allowed the detection of a factor, present in the high speed supernatant, which caused a dose-dependent inhibition of the binding of [³H]leu-enk to the morphine-sensitive and insensitive binding components. Investigations into the nature of the morphine-insensitive binding component demonstrated that it was an artifact since it was not detectable when bound and free ligand were separated by centrifugation. Furthermore, [³H]leu-enk bound to Whatman glass fiber filters, used to collect bound ligand, in a morphine-insensitive manner, and under conditions where the binding of [³H]leu-enk to the morphine sensitive component diluted proportionally with serial dilutions of the membranes, the binding to the morphine-insensitive component did not. The factor present in the high speed supernatant did not dialyze and its effects were mimicked by either trypsin or soybean trypsin inhibitor, but not by bovine serum albumin. The apparent inhibition of the binding of [³H]leu-enk to these binding components is probably not of biological significance, but the fact that the artifactual morphine insensitive binding component of striatal membranes has been shown to decrease by 20–30% following lesions of the substantia nigra suggests that the influence of this endogenous factor must be controlled for.     

DETECTION OF A SOLUBLE SEROTONIN-BINDING PROTEIN IN THE MAMMALIAN MYENTERIC PLEXUS AND OTHER PERIPHERAL SITES OF SEROTONIN STORAGE

Groups of neurons intrinsic to the mammalian myenteric plexus have been shown to have both tryptophan hydroxylase and a specific uptake mechanism for serotonin. They are probably serotonergic. A soluble protein with a high binding affinity for serotonin, similar to a protein previously found in rat brain by TAMIR & HUANG (1974), has now been found in the myenteric plexus of both rabbit and guinea pig. Partial purification of the protein from the rabbit's myenteric plexus by ammonium sulfate fractionation increased the ratio of specific to nonspecific serotonin binding almost 3-fold. Two dissociation constants for serotonin binding were obtained by equilibrium dialysis: 6.7 × 10^?10 M and 4.8 × 10^?7 M. The protein was similar to the soluble serotonin-binding protein of CNS: the indole derivatives 5, 6- and 5, 7-dihydroxytryptamine, and 6-hydroxytryptamine inhibited serotonin binding by 50% at 10^?7 M; norepinephrine was a poor inhibitor of serotonin binding; most of the serotonin-protein complex had a very high molecular weight and did not penetrate a 6.5% acrylamide gel. The appearance of the serotonin binding protein during development of the intestine in fetal rabbit correlates closely with the development of a serotonin uptake mechanism by nerves of this tissue and precedes the ingrowth of the adrenergic innervation. In-vitro administration of 6-hydroxydopamine to adult animals has no effect on the binding capacity for serotonin. Binding activity in denervated preparations is only 1/5 that of innervated tissue. It is concluded that the serotonin-binding protein, which has been found associated with serotonergic pathways in the CNS, is found associated with serotonergic neurons in the periphery as well. Since a similar serotonin-binding protein is also found in sheep thyroid, which stores but does not take up serotonin, the protein may be a component of the serotonin storage mechanism.     

THE BINDING OF [3H]MEPYRAMINE TO HISTAMINE H1 RECEPTORS IN GUINEA-PIG BRAIN

The promethazine-sensitive binding of [³H]mepyramine to a membrane fraction from guinea-pig whole brain is saturable with a dissociation constant of 1.7 × 10^-9M. The maximum amount of [³H]mepyramine binding varied widely between preparations, range 122–365 pmol/g protein, with a mean value of 227 ± 52 pmol/g protein. The inhibition of [³H]mepyramine binding by a number of drugs correlated closely with their potency as histamine H₁ antagonists. (+) Chlorpheniramine was 240-fold more potent as an inhibitor of [³H]mepyramine binding than (-)-chlorpheniramine. All antagonists inhibited the binding of [³H]mepyramine to the same extent, but the Hill coefficients characterising the inhibition curves did not all approximate to unity, the value expected for a simple antagonist-receptor equilibrium. The distribution of histamine H₁ receptors, defined by the promethazine-sensitive binding of [³H]mepyramine, in 11 different brain regions was uneven with the largest amounts in cerebellum, superior and inferior colliculus and hypothalamus and the smallest in caudate nucleus, brain stem and spinal cord.     

THE BINDING OF TRIETHYLTIN TO RAT BRAIN MYELIN

—A high affinity binding site for triethyltin was found in rat brain myelin with an affinity of approx 6·6 × 10⁵m ⁻¹ at pH 7·5. Competitive binding studies showed that triethyl-lcad had about the same affinity and trimethyltin 30 times lower affinity than triethyltin. Hexachlorophane and 3,5-diiodo-4′-chlorosalicylanilide did not prevent triethyltin binding to rat brain myelin. Since triethyltin, hexachlorophane and 3,5-diiodo-4′-chlorosalicylanilide all produce similar oedematous lesions in the brain of rats, whereas triethyl-lead and trimethyltin do not, it is concluded that the high affinity triethyltin binding site either is not involved or is not the only factor in oedema production.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A–II. MEASUREMENT OF THE TURNOVER RATES OF MAO A DURING ONTOGENESIS IN THE RAT BRAIN

The combined measurement of MAO A activity (using [³H]5-HT as a specific substrate) and [³H]harmaline binding capacity indicated that the concentration of MAO A in brain was higher in 14-28 day old rats than in adult animals. The turnover rates of this enzyme in the forebrain and the brain stem of young (14-28 day old) and adult rats were calculated by following the recovery of MAO A activity and of [³H]harmaline binding capacity after an acute treatment with pargyline (75mg/kg i.p.). Both the fractional rate constant for MAO A degradation and its synthesis rate per g of fresh tissue were significantly higher in young animals. However, the calculation of the absolute synthesis rates of MAO A per brain area gave very similar values in young and adult animals: 1.3-1.5 × 10¹³ molecules of MAO A synthesized per day in the forebrain and 2.3-2.9 × 10¹² molecules per day in the brain stern. The results illustrate the validity of using [³H]harmaline binding to evaluate possible changes in the turnover rate of MAO A in tissues.     

Chemico-biological interaction of Etravirine and its β-Cyclodextrin complex with macromolecular targets

The interaction of etravirine with β-cyclodextrin is analyzed by UV–visible absorption, infrared, fluorescence, nuclear magnetic resonance, two-dimensional rotational frame nuclear Overhauser effect spectroscopy, and molecular modeling studies. The 4-hydroxy-3, 5-dimethylbenzonitrile moiety is found to take part in the binding. The stoichiometry of the inclusion complex of ET with β-CD is 1:1 with the binding constant of 2.03 × 10³ mol^?1 dm³. The binding of ET with calf thymus DNA (ctDNA) and bovine serum albumin (BSA) protein is investigated in the presence and the absence of β-CD. Fluorescence enhancement is observed during the binding of ET with ctDNA in the absence of β-CD, whereas in the presence of β-CD, fluorescence quenching is observed. The binding constants of the binding of ET and ET–β-CD to ctDNA are 7.84 × 10⁴ and 4.38 × 10⁴ mol^?1 dm³, respectively. The binding constant of the binding of ET and ET–β-CD to BSA are 3.14 × 10⁴ and 1.6396 × 10⁴ mol^?1 dm³, respectively. The apparent binding constants between ET–β-CD complex and ctDNA or BSA protein decreases significantly. The numbers of binding sites of interaction of ET with BSA protein and the binding distance between BSA protein and ET the absence and the presence of β-CD differ. β-CD modulates the binding of ET with the macromolecular targets.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS

A high-affinity (K_d= 5.9 nM) specific binding site for [³H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [³H]serotonin ([³H]5-HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra-synaptosomal origin exhibited very similar properties with respect to [³H]harmaline binding characteristics and MAO A activity. Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [₃H]harmaline. Logit-log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [³H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 10²-2 × 10³ times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [³H]harmaline from its specific binding site. K_i and IC₅₀ values for the inhibition of [³H]harmaline binding by MAO I and MAO substrates (tryptamine, 5-HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [³H]5-HT as the substrate. In conclusion, the specific binding site for [³H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [³H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.     

COMPARISON OF THE BINDING’ OF β-ALANINE AND y-AMINOBUTYRIC ACID IN SYNAPTOSOMAL-MITOCHONDRIAL FRACTIONS OF RAT BRAIN1

Abstract— Na⁺-dependent ‘binding’ of β-alanine and GABA was examined with synaptosomal-mitochondrial fractions of rat brain incubated for 10 min at 0°C. GABA was bound to a much greater extent than β-alanine to particles of cerebral cortex, whole cerebellum and brain stem. For cerebral cortex, the binding capacity (B_max) for GABA was about 18 limes greater than that for β-alanine. and the affinity of the particles for GABA was about 2′ times greater than for β-alanine. The order of potency of GABA binding to brain regions was cerebral cortex > cerebellum > brain stem, whereas that for β-alanine was the reverse. If the binding of β-alanine is taken to indicate the glial component of the Na⁺-dependent binding process for GABA, then most of the GABA was bound to neuronal elements under the conditions employed.     

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.     

CHARACTERISTICS AND REGIONAL DISTRIBUTIONS OF TWO DISTINCT [3H]NALOXONE BINDING SITES IN THE RAT BRAIN

Using [³H]naloxone at a concentration of 4.5 nm , the potent opiate agonist etorphine as well as the potent antagonist diprenorphine displace only about 75% of specific naloxone binding P₂ fractions from rat whole forebrain, without additive effect. Several other opiates and antagonists completely displace specific naloxone binding. This indicates that etorphine and diprenorphine specifically bind to one and the same naloxone binding site (type I) while leaving another naloxone binding site (type II) unaffected. Type I binding sites are much more thermo-labile than type II. [³H]Naloxone binding to type I sites is unaffected by incubation temperature in the range 10 to 25°C. while binding type II sites decreases rapidly with increasing incubation temperature, no specific type II binding being detectable at or above 20°C. The two naloxone receptor types also differ with respect to pH dependence, and affinity for naloxone with types I and II having affinity constants (K_d) of 2 and 16 nm , respectively, at 0°C. The two binding sites have different regional distributions with high relative levels of type II receptors in cerebellum and low relative levels in pons-medulla and striatum. In whole rat brain there are about 4 times as many type II receptors as type I. These results suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor types which are probably not agonist/antagonist aspects of the same receptor.     

Binding of a chromen-4-one Schiff’s base with bovine serum albumin: capping with β-cyclodextrin influences the binding

This work deals with the synthesis of 6-methyl-3-[(4′-methylphenyl)imino]methyl-4H-chromen-4-one (MMPIMC), its binding to β-cyclodextrin, and the influence of the cyclodextrin complexation on the compound’s binding to bovine serum albumin (BSA). The 1:2 stoichiometry for the complexation of MMPIMC with β-cyclodextrin is determined with the binding constant of 1.90 × 10⁴ M^?2. The structure of host–guest complex plays a role in protein binding of MMPIMC. One- and two-dimensional NMR spectra are used to determine the mode of binding of the guest to β-cyclodextrin cavity and the structure of the inclusion complex is proposed. The binding of MMPIMC with BSA in the absence and the presence of β-cyclodextrin is studied. The binding strengths of MMPIMC–BSA (1.73 × 10⁵ M^?1) and β-cyclodextrin-complexed MMPIMC–BSA (9.0 × 10⁴ M^?1) show difference in magnitude. The Förster Resonance Energy Transfer efficiency and the proximity of the donor and acceptor molecules, are modulated by β-cyclodextrin. Molecular modeling is used to optimize the sites and mode of binding of MMPIMC with bovine serum albumin.     

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.     

Further studies on the specific interaction of S-100 protein with synaptosomal particulates.

Abstract— The specific interaction of S-100 protein with disrupted synaptosomes was further investigated. The specific binding is a saturable and reversible process, and is time, temperature, and strictly Ca²⁺ -dependent. Two affinities affect the interaction (K_ins= 7.04 × 10^?9 M. 1.28 × 10¹² binding sites/ mg protein; K_ins2= 3.91 × 10^?7M, 2.96 × 10¹³ binding sites/mg protein). The half-saturation time is about 5.5 min at 37°C. The half-life of the complex is 17 min at 37°C. At 0°C the binding is 75% slower than at 37° C, and only one-third of the binding sites are involved. The binding capacity is decreased by high NaCl concentrations and by pretreating membranes at high temperatures. Digestion of membranes with trypsin practically abolishes the specific binding. Treatment of membranes with phospholipase C decreases the specific binding, while phospholipase D enhances it to some extent. Other lipid extractors decrease significantly the extent of the interaction. Synaptic plasma membranes seem to be the synaptosomal component involved in the high affinity binding. The S-100 binding activity seems to undergo developmental changes, the adult values of kinetic parameters being reached around the 16th postnatal day in the rat. The results are discussed also in relation to the membrane-bound fraction of S-100.     

A characterization of alpha-bungarotoxin binding in the brain of the horseshoe crab, Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin to membrane fragments prepared from Limulus brain tissue has been investigated. Toxin binding approaches saturation in the range of 30 to 40 nm, with maximum binding of 2 to 6 pmol/mg of protein. The saturation kinetics and the rate of displacement of bound toxin are consistent with multiple toxin binding sites. Pharmacological studies show that binding is inhibited by both cholinergic agonists and antagonists, I₅₀′s for inhibition by d-tubocurarine, nicotine, decamethonium, carbachol, and atropine are 2 × 10^?6, 7 × 10^?6, 2 × 10^?5, 6 × 10^?4, and 3 × 10^?4m, respectively. Nicotinic ligands inhibited binding much more effectively than muscarinic ligands. Toxin binding activity was solubilized with Triton X-100. Velocity sedimentation analysis of the solubilized activity revealed three separate components. Seventy to eighty percent of the binding activity had a sedimentation coefficient of 8.6 S. The remaining activity was composed of two components with sedimentation coefficients of 15.1 and 17 S.     

THE CATION SENSITIVITY OF THE ACETYLCHOLINE RECEPTOR FROM TORPEDO CALIFORNICA

—The effects of various cations and cholinergic ligands on the rate of α-bungarotoxin-acetylcholine receptor complex formation has been studied by means of a DEAE-cellulose disk assay technique. The reaction rate is reduced to one half the initial rate in the presence of 2·5 · 10^?6m acetyleholine. a value close to the observed K_D of ACh measured by equilibrium dialysis. Inhibition constants of about 5 mM were obtained for most monovalent cations whereas divalent cations gave inhibition constants of 0·05-0·2 mm . The rate and extent of toxin-receptor complex formation was also investigated as a function of hydrogen ion concentration; the rate of formation reaches a maximum at pH 7·5 and a group with a pK about 6 inhibits toxin binding to the receptor when protonated. These data can be correlated with the observed effects of inorganic cations on the binding of cholinergic ligands in vivo at the neuromuscular junction. Given the affinities of the individual cations, it is possible to predict how the apparent affinity of a cation-sensitive cholinergic ligand will change with variations of buffer composition.     

NOVEL CLASS OF DNA BINDING MOTIFS BASED ON BISTETRAHYDROFURAN AND BISFURAN SKELETON WITH LONG ALKYL CHAINS

Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this paper, new DNA binding molecules; diamino-bistetrahydrofuran (bisTHF) and diamino-bisfuran are reported. The bisTHF ligand with RR configuration at the amino groups and C8 alkyl chains (RR8) stabilized GC-rich duplex. In contrast, bisfuran compounds stabilized AT-rich duplex. The binding affinity of RR8 with 12 mer duplex DNA was determined by isothermal titration calorimetry to be 3.3 × 10⁸ M^?1.     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.