Gamma-aminobutyric acid, bicuculline, and post-synaptic binding sites

The binding of γ-aminobutyric acid (GABA) to synaptosomal fractions of the rat cerebellar cortex has been examined at 0–4°C in the presence and absence of bicuculline, chlorpromazine, and/or Na⁺. A GABA-binding component has been demonstrated in the synaptosomal fraction which is competitively inhibited by bicuculline. In addition, this binding component persists in the absence of Na⁺ and in the presence of chlorpromazine. It seems likely that this binding component is the post-synaptic binding site or “receptor” of GABA.     

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 $\text{in vitro}$.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Effects of chaotropic anions on bicuculline inhibition of high affinity γ-aminobutyric acid binding in bovine retina and rat brain membranes

Chaotropic anions (ions that favour transfer of apolar groups to water) increased bicuculline inhibition of ³H-γ-aminobutyric acid binding to bovine retinal membranes as previously reported for rat forebrain membranes. The increased bicuculline inhibition was reversible when the chaotropic anion was removed thus ruling out the possibility that ‘endogenous regulators’ were being removed by the chaotropic anions. Another possible explanation for the enhanced bicuculline inhibition is an increase in the solubility of bicuculline, an organic compound that is sparingly soluble in water; however, when bicuculline-methiodide, a more water soluble form of bicuculline was used, no difference was noted in this enhancement.Although the chaotropic anions temporarily increase the bicuculline inhibition of γ-aminobutyric acid binding, they do not increase ³H-γ-aminobutyric acid receptor binding as previously suggested. Thiocyanate or perchlorate have no effect on ³H-γ-aminobutyric acid receptor binding to rat forebrain or cerebellar membranes. Although thiocyanate slightly inhibits γ-aminobutyric acid receptor binding to bovine retinal membranes, perchlorate has no effect. The previous observation that sodium perchlorate enhanced γ-aminobutyric acid binding in bovine retina was due to the enhancement of sodium-dependent binding to a nipecotic acid-sensitive binding site (perhaps an uptake site).The mechanism of action of chaotropic anions in vitro on γ-aminobutyric acid binding may be through an alteration in the interaction of the receptor with the antagonists, bicuculline or bicuculline-methiodide, but is not through the removal of a component that blocks the binding site or regulates the receptor's properties, as evidenced by the reversibility of the chaotropic anion effect and the lack of effect on agonist binding.     

Glucocorticoid tight binding in rat liver and thymus particles

Glucocorticoid binding to certain cell particles of rat liver and thymus following treatments $\text{in vivo}$ and $\text{in vitro}$ consists in part of a very “tight binding” that resists hot and cold perchloric acid extractions. This binding is found in thymus nuclei and in liver cytoplasmic particles, but not in liver nuclei nor in thymus mitochondria or microsomes. The existence of “tight binding” coincides with the ability of the same particles to bind free corticoid directly in incubations $\text{in vitro}$. The difference in the cellular location of this binding suggests that different methods of glucocorticoid activation exist in the anabolic target tissue, liver, and the catabolic target tissue, rat thymus.     

Effects of progesterone on the binding of estradiol-receptor to rabbit uterine chromatin

Three day progesterone treatment of ovariectomized rabbits increased $\text{in}$$\text{vitro}$ uterine estrogen-receptor binding to uterine chromatin. The increased binding was traced to changes in chromatin but not the cytosol. Both the number of chromatin acceptor sites and the binding affinity were higher in treated animals. Furthermore, chromatin acidic protein to DNA ratios from treated rabbits were higher by approximately the same factor as for binding sites. A mechanism of synergistic interaction is suggested.     

D-LSD binding to brain homogenates: possible relationship to serotonin receptors

Using a rapid filtration technique we find stereospecific high-affinity $\text{d}$-LSD binding (4 × 10^?9M, half-saturation) in brain fractions from a number of subcortical as well as cortical brain regions. Among the putative neurotransmitters tested only serotonin effectively displaces $\text{d}$-LSD from this specific binding site. Moreover, only the serotonin-displaceable component of binding is saturable in a high-affinity range. No change is observed in specific $\text{d}$-LSD binding in forebrain homogenates from rats in which ascending serotonergic pathways are destroyed by lesions in the raphe nucleus. We conclude that a vast majority of the $\text{d}$-LSD binding sites may be associated with a postsynaptic serotonin receptor rather than a presynaptic receptor associated with serotonergic (raphe) inputs.     

Receptor-like stereospecific binding of a pyrethroid insecticide to mouse brain membranes

A heterogeneous particulate fraction of mouse brain homogenates binds NRDC 157 (3-phenoxybenzyl [1$\text{R}$,$\text{cis}$]-3-(2,2-dibromovinyl)-2,2- dimethylcyclopropanecarboxylate), a potent pyrethroid insecticide, stereospecifically and with high affinity. Stereospecific binding is a minor component of total binding (2.8%); the remainder of observed binding is predominantly nonspecific and unsaturable. Stereospecific binding is half-saturated at 4×10^?8$\text{M}$ and fully saturated at concentrations in excess of 1×10^?7$\text{M}$. The stereospecific binding capacity of this preparation was 200–250 pmoles of NRDC 157 per gram equivalent of brain tissue (2.3–2.8 pmol/mg protein). This binding site may represent the neural receptor involved in the stereospecific toxic action of pyrethroids.     

Met-tRNAfMet binding in murine dystrophy

Dystrophic mice of the C57B1 $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ strain and of the ReJ 129 $\text{dy}\text{dy}$ strain and littermate controls were used to prepare met-tRNA^fMet binding factors. The tissues were homogenized and fractions were obtained which contained ribosomes. The binding factors were assayed by the binding of [³⁵S]methionyl-tRNA to control liver ribosomes. The binding, i.e. eukaryotic initiation factor 2 (elF 2) activity, was measured in brain, liver and muscle and in all of these tissues there was a significant decrease in the dystrophic mice. This decrease in initiation factor activity of hindleg muscle resembled, in the direction of the effect, the decrease in elongation factor activity of hindleg muscle resembled, in the of $\text{dy}\text{dy}$ mice previously reported by our laboratory. Thus these two defects, taken together may help to explain the marked wasting of the muscles. The decrease in brain in both strains provides evidence for nervous tissue involvement in genetic dystrophy.     

Cooperativity in ligand binding: a new graphic analysis.

When analyzing binding of ligands to macromolecules, the existence of site-site interactions complicates a straightforward interpretation of the binding parameters obtained through classical analytical methods, such as the Scatchard plot. For describing site-site interactions, we propose a new parameter, the $\text{average affinity}$ of the receptor sites, $\text{K}$, calculated as $\text{(}\text{B}\text{F}\text{)/(R}{}_{\mathrm{o}}\text{?B)}$. Plotting $\text{K}$ as a function of fractional occupancy ($\text{B}\text{R}{}_{\mathrm{o}}$), reveals that: (1) at very low occupancy a limiting high $\text{K}$ is obtained ($\text{K}$_e) (“empty sites” conformation); (2) when the fraction of sites filled increases above a certain threshold, $\text{K}$ begins to fall due to increasing site-site interactions until (3) a limiting low $\text{K}$ ($\text{K}$_f) is obtained (“filled sites” conformation). This method has been successfully applied to the negative cooperativity of insulin receptors.     

The effect of enzyme induction on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes

The effect of pretreatment with phenobarbitone, rifampicin, β-naphthoflavone, antipyrine and spironolactone on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes has been examined and the corresponding changes in microsomal P-450 content and cytochrome $\text{c}$ reductase activity measured. Rifampicin produced the greatest increase (220%) in irreversible binding while phenobarbitone produced the greatest increase in both microsomal P-450 content (172%) and cytochrome $\text{c}$ reductase activity (210%). There was no correlation of irreversible binding with either microsomal P-450 content or with cytochrome $\text{c}$ reductase activity.     

Demonstration of specific "high affinity" binding sites for [3H] imipramine on human platelets

High affinity and saturable binding sites for [³H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [³H] imipramine binding. The binding of [³H] imipramine to human platelets is of high affinity $\text{(}\text{Kd}\text{? 1.4}\text{nM}\text{)}$, saturable $\text{(}\text{Bmax}\text{? 625}\text{fmols/mg prot}\text{)}$, and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [³H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.     

A mitochondrial defect in brown adipose tissue of the obese (ob/ob) mouse: reduced binding of purine nucleotides and a failure to respond to cold by an increase in binding.

Atractyloside-insensitive binding of purine nucleotides is reduced in brown adipose tissue mitochondria of the obese ($\text{ob}\text{ob}$) mouse. Exposure of the $\text{ob}\text{ob}$ mouse to 4°C does not induce the usual increase in binding. Atractyloside-insensitive binding of purine nucleotides is believed to be a measure of the heat-producing proton conductance pathway in brown adipose tissue mitochondria. It is, therefore, suggested that the impaired thermogenesis of the $\text{ob}\text{ob}$ mouse is due to a defect in this pathway in the mitochondria of the brown adipose tissue, the major thermogenic tissues in rodents. The greater metabolic efficiency which would result from a reduced operation of this pathway might be the basis for the obesity in the $\text{ob}\text{ob}$ mouse.     

Evidence for cooperative Mn-ATP binding with Bacillus sp. glutamine synthetase

The binding of Mn-ATP with $\text{B. subtilis}$ glutamine synthetase, observed kinetically at 37°, pH 7.0, is cooperative (Hill $\text{n}\text{= 2.3, S}{}_{\mathrm{0·5}}\text{= 0.36mM)}$, a phenomenon overlooked in earlier studies. The Arrhenius plot is biphasic with a break at 26°C. Similar behavior is observed with the thermophilic $\text{B. stearothermophilus}$ enzyme, but is absent with the enzymes from $\text{E. coli}$, plant, and mammaliam sources under optimal assay conditions. The temperature dependence of the intrinsic fluorescence of the protein is also non-linear, and the intersection point of 18° shifts to 30° upon binding of substrates. These results are interpreted as indicating that $\text{Bacillus sp}$. enzymes can assume multiple, functionally important conformational states related to Mn-ATP binding at 37°. They also emphasize further that critical differences in mechanism exist among glutamine synthetases from different sources.     

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.     

Partial purification and properties of a non-ligandin (3H) 3-methylcholanthrene binding protein from liver cytosol

(³H) 3-Methylcholanthrene binds $\text{in vivo}$ to a macromolecule in addition to the previously reported binding to ligandin in liver cytosol. The properties of this second molecule are identical to those of the glucocorticosteroid receptor (Binder II) through 400 fold purification over the cytosol proteins (elution position from DEAE-Sephadex A-50 columns, molecular weight by gel filtration and pI value by isoelectrofocusing). The carcinogen, probably a metabolite, binds very strongly or covalently to the macromolecule $\text{in vivo}$, but non-covalently $\text{in vitro}$ in the absence of microsomes. Large amounts of unlabeled carcinogen administered $\text{in vivo}$ do not compete significantly with subsequent (³H) dexamethasone binding to the hormone receptor fraction $\text{in vitro}$. Methylcholanthrene and dexamethasone do not compete for binding sites $\text{in vitro}$ on isolated unlabeled Binder II leading to the conclusion that the glucocorticosteroid receptor and the methylcholanthrene binding protein are distinct entities.     

The effect of sodium ion on antinociception and opiate binding in vivo.

Large quantities of NaCl and CaCl₂ but not KCl given intrapertoneally decreased the antinociceptive activity of morphine. NaCl also antagonized the effect of morphine on the stereo-specific binding of opiates. This high dose of NaCl doubled the level of sodium in the brain but did not alter the specific gravity of brain tissue. These $\text{in}$$\text{vivo}$ effects of NaCl confirm the antagonistic effects of NaCl $\text{in}$$\text{vitro}$ that have been reported.     

pH dependence of magnesium ion binding to prothrombin fragment 1 and gamma-carboxyglutamic acid-containing peptides via 25Mg NMR.

The binding of magnesium ions to two tripeptides, $\text{L}$-Arg-$\text{D}$-Gla-$\text{D}$-Gla-OMe and Z-$\text{L}$-Arg(NO₂)-$\text{D}$-Gla-$\text{D}$-Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by ²⁵Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.     

Lac repressor binding to poly (d(A-T)). Conformational changes

The binding of $\text{lac}$ repressor to poly [d(A-T)] at low ionic strength has been investigated by circular dichroism, fluorescence and light scattering. Poly [d(A-T)] undergoes an important conformational change upon binding to $\text{lac}$ repressor. The maximum number of binding sites corresponds to about one tetrameric repressor per 11 base pairs of poly[d(A-T)]. The inducer isopropyl β-D-thiogalactoside (IPTG) does not affect the binding of $\text{lac}$ repressor to poly[d(A-T)]. It binds equally well to free and poly[d(A-T)] -bound repressor.     

On the possibility of carnitine binding within the brown adipose tissue cell

Even though injected radioactive carnitine is found to accumulate in brown adipose tissue of suckling rats, no consistent specific binding to a protein in the high speed supernatant of this tissue could be demonstrated, either $\text{in vivo}$ or $\text{in vitro}$. On $\text{in vitro}$ incubation of $\text{in vivo}$ prelabelled brown fat, 80% of the label was released into the medium within 20 minutes.