Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background

We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (K_d) and gp120-to-CD4 inhibition constants (K_i). The PA analogs were selected to satisfy Lipinski''s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity.

Principal Findings

PA analog 2-bromopalmitate (2-BP) was most efficacious with K_d ∼74 nM and K_i ∼122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher K_d ∼140 nM and K_i ∼354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with K_d ∼364 nM and inhibiting gp120-to-CD4 binding with K_i ∼1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry.

Significance

Considering observed differences between K_i and K_d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry.     

Two binding sites in acetylcholine receptor from Torpedo marmorata electroplax

The sensitivity of acetylcholine receptor to eleven cholinergic drugs, phospholipase A, heat and pH provided evidence that the so-called high-affinity binding (K_d for acetylcholine 11 nm in 1% Triton) and low-affinity binding (K_d 562 nm) were related to two distinct binding sites. The low-affinity binding site was less sensitive to heat and several of the cholinergic drugs, but was a little more sensitive to bungarotoxin than the high-affinity site. Zinc (0.4 mm) and EDTA (10 mm) abolished acetylcholine binding to both sites; the EDTA inhibition was time-dependent.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1)

The binding of various nucleotides to chloroplast coupling factor CF₁ was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 μM added MgCl₂, the protein has a single strong site/mol protein with K_d = 0.5 μM for ADP and high specificity (K_d > 20 μM for ?ADP, GDP, CDP). In the presence of 5 mM MgCl₂, the protein has two independent tight ADP sites (K_d = 0.4 μM) of low specificity (K_d ≈ 0.8, 2, and 2 μrmM, respectively for ?ADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence

Metals are thought to play a role in the structure of many viruses. The crystal structure of the T=3 icosahedral cowpea chlorotic mottle virus (CCMV) suggests the presence of 180 unique metal-binding sites in the assembled protein cage. Each of these sites is thought to involve the coordination of the metal by five amino acids contributed from two adjacent coat protein subunits. We have used fluorescence resonance energy transfer (FRET), from tryptophan residues proximal to the putative metal-binding sites, to probe Tb(III) binding to the virus. Binding of Tb(III) was investigated on the wild-type virus and a mutant where the RNA binding ability of the virus was removed. Tb(III) binding was observed both in the wild-type virus (K_d=19 M) and the mutant (K_d=17 M), as monitored by the increase in Tb(III) fluorescence (545 nm) and concomitant decrease in tryptophan fluorescence (342 nm). Competitive binding experiments showed Ca(II) to have about 100-fold less affinity for the binding sites (K_d=1.97 mM). This is the first direct evidence of metal binding to the putative metal-binding sites, originally suggested from the crystal structure of CCMV.     

High-affinity phlorizin binding to brush border membranes from small intestine: Identity with (a part of) the glucose transport system,dependence on the Na+-gradient,partial purification

In the presence of an NaSCN gradient phlorizin binds with a high affinity (K_d ? 4.7 μM) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for K_d corresponds closely to that of K_i determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min). The high affinity sites are related to glucose transport based on the similarity of the K_d and K_i values under a variety of conditions and on the inhibition of the binding by D-glucose but not by D-fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2–3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein.     

Alpha2 adrenergic receptors are located prejunctionally in the Auerbach's plexus of the guinea pig small intestine: Direct demonstration by radioligand binding

We provide direct evidence that alpha₂-receptors in the guinea pig small intestine are localized prejunctionally in neurons of the Auerbach's plexus. The alpha₂-agonist ligand [³H]clonidine bound to a single saturable class of sites with a K_d of 1–2 nM and a capacity of approximately 70 fmol/mg protein in membranes from the innervated longitudinal and circular muscle layers of the intestine. By a special dissection technique the Auerbach's plexus could be completely removed from the longitudinal muscle. In these denervated preparations the clonidine binding sites were virtually completely removed whereas the expected binding was observed in innervated controls. The innervated preparations also contained a small number of alpha₁-receptors as revealed by binding with [³H]prazosin (capacity approximately 18 fmol/mg protein with a K_d of 0.4-0₃7 nM). Thus, the present study suggests that alpha₂-receptors ([³H]clonidine binding sites) are localized in neurons (i.e., prejunctionally) in the Auerbach's plexus of the guinea pig small intestine.     

Binding of a thromboxane A2/prostaglandin H2 agonist [H]U46619 to washed human platelets

The binding characteristics of [³H]U46619 to washed human platelets were studied. [³H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a K_d of 11 ± 4 nM (n=4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a K_d of 20 ± 7nM and a B_max of 9.1 ± 2.3 fmole/10⁷ platelets (550 ± 141 binding sites per platelet) (n=4). A number of compounds that act as either agonists or antagonists of the TXA₂/PGH₂ receptor were tested for their ability to inhibit the binding of [³H]U46619 to washed human platelets. The K_ds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [³H]U46619 binds to the putative human platelet TXA₂/PGH₂ receptor.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

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.     

Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors

The binding of TNP-ATP (2 or 3-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant K_d = 0.2 µM) and one with lower affinity (K_d = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (K_d = 3.6 µM). The binding of [³⁵S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (K_d = 7.5 µM) and six at the liver enzyme (K_d = 12 µM). The Paracoccus enzyme had only one binding site (K_d = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H⁺/e^- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H⁺/e^- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H⁺/e^- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

Probing of the porcine serum lipoprotein surfaces by Mn(II) binding: an e.s.r. study

Mn(II) ions were used for probing the surfaces of porcine LDL₁, LDL₂ and HDL. From the intensity of the e.p.r. lines corresponding to the unbound Mn(II) the percentage of the ions bound to the lipoprotein surface is determined. From the titration curves the binding parameters, dissociation constant. K_d, and the number of binding sites, n, in all the three lipoproteins studied have been derived. There are at least two types of binding sites in each lipoprotein class. The ”weak’ binding sites are charaterized by approximately the same value of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(≈ 6.2 × 10}{}^{\mathrm{?3}}\text{mol l}{}^{\mathrm{?1}}$ and different values for n (n = 114 for LDL₁, n = 135 for LDL₂ and n = 28 for HDL). Similarly, for the ”strong’ binding sites K_d ≈ 1.6 × 10^?4 mol l^?1 and the number of binding sites is 15, 20 and 5 for LDL₁, LDL₂ and HDL respectively. It is concluded that the binding sites are probably located in the protein part of the lipoproteins and that they are mainly associated with the negatively charged amino acids.     

Interacalation of ethidium ion into DNA and RNA oligonucleotides

The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA₅G + rCU₅G and the analogous deoxyribo-oligonucleotides dCA₅G + dCT₅G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is ?14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 10⁵M^?1 for the two strong sites (4 × 10³M^?1 for the weak site) or K = 2.5 × 10⁵M^?1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 10⁴M^?1).     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts

A method is described for the determination of binding capacity (R_o) and dissociation constant (K_d) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [³H]dihydrotestosterone³ (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (R_o = 12,105 ± 4305 (S.D.) sites/cell; K_d = 1.0 ± 0.7 (S.D.) × 10^?9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.     

n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites

We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (K_d?=?40?μM) with a lower affinity than SDS (K_d?=?2?μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.     

Localization and pharmacological characterization of nicotinic-cholinergic binding sites in cockroach brain using α- and neuronal bungarotoxin

[¹²⁵I]α-Bungarotoxinisusedasaprobetostudythenicotinic-cholinergicreceptorinmembrane preparations of the cockroach brain. Binding is restricted mainly to particulate fractions of brain homogenates, is time dependent and is saturable above 2 nM with very low non-specific binding. Scatchard analysis indicates that binding is associated with a single affinity site (K_d = 1.09 nM) having a B_max of 8926 fmol/mg protein which is the highest concentration of binding sites yet reported in insects. Association kinetics are best fit by a mono-exponential model with a k_obs = 4.37 × 10⁻³s⁻¹. Dissociation is best described by a bi-exponential model giving dissociation constants of 1.18 × 10⁻⁵ and 9.94 × 10⁻⁵s⁻¹. The K_ds calculated from kinetic data are 0.029 and 0.25 nM suggesting the possibility of heterogeneous binding sites not detected by saturation studies. Displacement studies indicate that binding follows a nicotinic pharmacology and demonstrate the high affinity of methyllycaconitine and the anthelmintics, morantel and pyrantel. Displacement by neuronal bungarotoxin shows the presence of two distinct binding sites not differentiated by α-bungarotoxin. Autoradiographic studies show α-bungarotoxin to be binding to neuropile regions of the brain, to be displaced from these regions by agents effective in binding studies and demonstrate that the neuronal bungarotoxin binding sites can be regionally localized.