Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

The alteration of serotonin binding sites in aged human brain

The $\text{in}$$\text{vitro}$ binding of [³H]serotonin ([³H]5-HT) to cerebral cortex from young and old adult humans was studied. With cortex from human males 23–29 years old, the binding of [³H]5-HT was a saturable process, and bound [³H]5-HT could be displaced by unlabeled 5-HT or by lysergic acid diethylamide (LSD). Two separate binding sites were discernible by Scatchard analysis. The dissociation constants were 7 nM (Kd₁) and 52 nM (Kd₂), and the total number of binding sites were 0.65 (n₁) and 2.06 (n₂) pmoles/mg protein, respectively. In cerebral cortex from aged humans (61–70 years old), the dissociation constant for [³H]5-HT binding was 198 nM, and the total number of binding sites were 4.78 pmoles/mg protein. The alteration of serotonin binding sites may be related to cerebral malfunction in old people, particularly to mental depression and sleep disturbances that commonly occur.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

beta-Endorphin: characteristics of binding sites in the rat brain.

Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using $\text{[}{}^3\text{H-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the ligand. The binding is time dependent and saturable with respect to β_h-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of β_h-endorphin, is presented.     

Benzodiazepine binding in human brain: characterization using [3H]flunitrazepam.

[³H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [³H]flunitrazepam. The dissociation constant (K_D) of [³H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [³H]-flunitrazepam binding $\text{in}$$\text{vitro}$ correlated well with their potency in several $\text{in}$$\text{vivo}$ human and animal tests. The density of [³H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.     

Identification and characterization of angiotensin II receptors in cardiac sarcolemma

We have used [¹²⁵I] angiotensin II to investigate the presence of specific angiotensin II receptors in beef heart sarcolemmal membranes. The observed binding is saturable, reversible and specific. The apparent equilibrium dissociation constant is 2.23 ± 0.15 ($\text{x}$ ± SEM) and the maximal number of binding sites per mg membrane protein is 32.8 ± 5.4 fmol ($\text{x}$ ± SEM). The specific binding is 80–100% of the total [¹²⁵I] angiotensin II bound and is directly proportional to membrane protein concentration over the range of 33–173 μg protein per ml. Angiotensin II and its antagonists competed for binding in a potency order of (agent, K_i): angiotensin II, 0.9nM > Sar¹ Ala³, 7 nM > Sar¹-Ile³, 51 nM > Sar¹-Leu³, 427nM > angiotensin I, 1709 nM. The ability to characterize and quantify these receptors should now provide a method for investigating the mechanisms underlying the effects of angiotensin II on myocardial tissues.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.     

Imipramine binding site Temperature dependence of the binding of 3H-labeled imipramine and 3H-labeled paroxetine to human platelet membrane

The characteristics of ³H-labeled imipramine and ³H-labeled paroxetine binding to human platelet membranes were determined at various temperatures between 0 and 37°C. Both paroxetine and imipramine probably bind to the same molecular complex in the platelet membrane, but the binding characteristics are different for the two molecules. The dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) for imipramine increases from 0.3 nM to 7.0 nM with increasing incubation temperature in a continuous way, whereas $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for paroxetine is almost constant, about 0.05 nM, between 0 and 19°C, and first begins to increase from 0.06 nM to 0.16 nM between 20 and 37°C. This suggests that the binding of paroxetine to the binding site induces a conformational change in the molecular complex of the binding site, whereas the binding of imipramine takes place without conformational changes in the binding site.     

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.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (K_d between 15 and 450 nM) and different off-rates of the cAMP-receptor complex ($\text{t}\text{1}\text{2}$ between 0.7 and 150 s). The association of cAMP to the receptor and the dissociation of the cAMP-receptor complex still occur in the presence of 3.4 M ammonium sulfate. However, these processes are strongly altered. (1) Low concentrations of ammonium sulfate (≈ 50 mM) induce an approx. 2-fold increase of the number of cAMP binding sites. The same effect is induced by millimolar concentrations of CaCl₂. Ammonium sulfate and CaCl₂ are not additive, which suggests that these salts may act via the same mechanism. (2) High concentrations of ammonium sulfate (3.4 M) induce an alteration in the proportioning of the various cAMP binding sites to the components with the highest affinity. (3) High concentrations of ammonium sulfate (3.4 M) retard the dissociation of all binding sites about 3–6-fold, thus giving rise to an increase in the affinity of all cAMP-binding components.     

Binding characteristics of a potent enkephalin analog

A radioiodinated form of the highly potent enkephalin analog FK 33-824 has been characterized with respect to its binding properties $\text{in vitro}$. ¹²⁵I-FK 33-824 is distinctive among the short opioid peptides in three ways. First, ¹²⁵I-FK 33-824 binds stereospecifically to rat brain homogenates with very high affinity (K_d = 0.42 nM). Secondly, dissociation of the ¹²⁵l-labelled peptide from membrane-bound opiate receptors occurs with a relatively long $\text{τ}\text{1}\text{2}$ of 25 min at 4° in contrast to other enkephalins which dissociate more rapidly. Third, competitive binding analyses reveal that the ¹²⁵l-FK 33-824 binds equally well to both enkephalin (δ) and morphine (μ) classes of opiate receptors. These characteristics distinguish the ¹²⁵l-labelled peptide as a particularly suitable probe for molecular studies and purification of the opiate receptor.     

Temperature-sensitive high affinity [3H]serotonin binding: Characterization and effects of antidepressant treatment

Characterization of temperature-sensitive [³H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S₁ and S₂ receptors. $\text{In vivo}$ pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [³H]5-HT site, although [³H]ketanserin (S₂) densities were decreased by 50%. This suggested that possible S₂ components of [³H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC₅₀ = 3 nM) for a portion of the 1 nM Kd [³H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [³H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after $\text{in vitro}$ incubation. The complex inhibition data may therefore represent indirect interactions through another site.     

β-Endorphin: Characteristics of binding sites in the rabbit cerebellar and brain membranes

Specific binding of human β-endorphin to rabbit cerebellar and brain membranes was measured using $\text{[}{}^3\text{H}{}_2\text{-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the primary ligand. In both tissues binding was time dependent and saturable, with apparent equilibrium dissociation constants of 0.275 nM and 0.449 nM in the cerebellum and brain, respectively. The binding capacity of cerebellum is greater than that of brain. Kinetic studies showed that the association rate constants were 2.7 × 10⁷ M^?1min^?1 for cerebellum and 2.4 × 10⁷ M^?1min^?1 for brain. Dissociation of tritiated β_h-endorphin from both cerebellum and brain is not consistent with a first order decay from a single site. In the cerebellum, these is a time-dependent increase in slowly dissociating complex. The potency of several opioid peptides and opiates to inhibit the binding of tritiated β_h-endorphin was determined. Ligands with preference for μ, δ, and κ opiate receptor (morphine, Metenkephalin and ethylketocyclazocine) all have similar affinities toward β_h-endorphin sites in both brain and cerebellar membranes.     

Identification of alpha adrenoceptor subtypes in dog arteries by (3H) yohimbine and (3H) prazosin

Binding of the alpha adrenergic antagonists (³H) prazosin and (³H) yohimbine to membranes of dog arteries exhibit the characteristics expected of alpha adrenoceptors. Binding of both ligands is saturable with dissociation constants of 0.19nM and 1.15nM for (³H) prazosin and (³H) yohimbine respectively. A series of catecholamines inhibit binding of both ligands with a potency in the order $\text{epinephrine}\text{>}\text{norepinephrine}\text{a}\text{?}\text{isoproterenol}$, corresponding with the activity of these agents at alpha adrenoceptors in blood vessels. Competition for binding in both instances is stereoselective. ?-Phenylephrine has similar potencies in inhibiting (³H) prazosin and (³H) yohimbine specific binding whilst the imidazoline related partial alpha adrenergic agonists clonidine and guanfacine are more potent in inhibiting (³H) yohimbine specific binding. The affinity of prazosin for the (³H) yohimbine binding site is approximately 2500 times less than for the (³H) prazosin site whilst yohimbine is approximately 150 times more potent in inhibiting (³H) yohimbine than (³H) prazosin specific binding. Non-selective alpha adrenergic antagonists have similar affinities for both binding sites. The concentrations of (³H) yohimbine binding sites in different arteries vary about two fold whilst for (³H) prazosin the variation was about three fold. These results indicate that there are two discrete noradrenergic binding sites in the major arteries of dog which have binding properties expected of alpha₁ and alpha₂ adrenoceptors.     

Stimulation of respiration-linked proton efflux in Escherichia coli by carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP).

The proton efflux from intact, anaerobic $\text{Escherichia}\text{coli}$ cells following a small oxygen pulse is both slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>M</mtext><mtext>2</mtext>}}\text{～-10s}$) and inefficient ($\text{H}{}^{\mathrm{+}}\text{O}\text{～-0.5}$. Very low levels (<80 nM) of the proton ionophore carbonylcyanide-$\text{p}$-trifluoromethoxyphenylhydrazone (FCCP), which have no detectable effect upon active transport, cause a 3–5 fold stimulation in the extent of proton efflux without affecting the efflux rate. At slightly higher concentrations of FCCP (80 nM to 0.5 μM), a sharp inhibition of this increased proton efflux occurs, with the $\text{H}{}^{\mathrm{+}}\text{O}$ ratio obtained in the presence of 0.5 μM FCCP approximately equal to that obtained in the absence of FCCP. Still higher concentrations of FCCP (> 1 μM), which inhibit active transport, cause a further gradual decrease in the $\text{H}{}^{\mathrm{+}}\text{O}$ ratio. The unusual increase in the apparent efficiency of H⁺ efflux by <80 nM FCCP is not accompanied by an increase in the rate of membrane deenergization following an O₂ pulse, although such an increase is seen with the higher (uncoupling) FCCP concentrations.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Inhibition of cyclopropane fatty acid synthase by sinefungin and A9145C

Sinefungin and A9145C, antifungal antibiotic analogs of S-adeno-sylmethionine isolated from $\text{Streptomyces}\text{,}\text{griseolus}$, have been found to be very effective $\text{in}\text{,}\text{vitro}$ inhibitors of cyclopropane fatty acid synthase from $\text{Lactobacillus}\text{,}\text{plantarum}$. Both compounds exhibit linear competitive inhibition with a K_i for Sinefungin of 220 nM and a K_i for A9145C of 11 nM.     

Na+-independent binding of GABA to the triton X-100 treated synaptic membranes from cerebellum of rat brain

The treatment of the membranes from cerebellum of rat brain with 0.5% Triton X-100 increases both the affinity and the density of the Na⁺-independent binding sites for ³H-GABA (γ-aminobutyric acid) from the values obtained from membranes of rat brain after an extensive freezing and thawing treatment (Young $\text{et al.}$, 1976). Upon repeated washings of the Triton-treated membranes, the binding of ³H-GABA is further increased and follows biphasic kinetics which indicates two binding components having dissociation constants of 5.9 and 27 nM and densities of 1.35 and 3.9 pmole/mg protein, respectively. GABA agonist, imidazoleacetic acid, and the GABA antagonists, bicuculline and d-tubocurarine, inhibit 50% of ³H-GABA binding at 1, 47 and 85 μM concentrations (IC₅₀ values), respectively. The IC₅₀ values for these compounds are unchanged by Na⁺. Thus, the Na⁺-independent binding of ³H-GABA to the Triton-treated membranes may represent binding to the synaptic GABA receptors.