Independent sites of low and high affinity for agonists on Torpedocalifornica acetylcholine receptor

It is demonstrated that two classes of binding site for acetylcholine are present on $\text{Torpedo}\text{californica}$ acetylcholine receptor. One class is the well documented site on each of the two subunits of 40,000 daltons, which can be covalently modified by bromocetylcholine. Both in the absence and in the presence of bromoacetylcholine another binding site is shown to exist by virtue of acetylcholine dependent fluorescence changes in the receptor covalently modified by 4-[N-(iodoacetoxy)ethyl-N-methyl]-amino-7-Nitrobenz-2-oxa-1,3 diazole (IANBD). This site has a low affinity for acetylcholine (K_d ～ 80 μM) that corresponds closely with the known concentration dependence of acetylcholine mediated activation of this receptor and we conclude that it may represent a site of association that participates in channel opening in this system.     

A stereoselective pentobarbital binding site in cholinergic membranes from Torpedocalifornica

Acetylcholine receptor-rich membranes from $\text{Torpedo}\text{californica}$ contain a binding site for [¹⁴C] pentobarbital which has a dissociation constant of 210 ± 24 μM and 1.4 ± 0.18 sites per acetylcholine site. (+) pentobarbital competes for this site three times more effectively than (?) pentobarbital. Cholinergic ligands decrease [¹⁴C] pentobarbital binding and this effect is blocked by pre-incubation with α-bungarotoxin. Pentobarbital decreases [³H] acetylcholine binding non-competitively with an apparent dissociation constant similar to the dissociation constant for [¹⁴C] pentobarbital binding. Thus, the pentobarbital and acetylcholine binding sites appear to interact with each other allosterically.     

Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment

The regulation of ligand binding to the muscarinic acetylcholine receptor in developing chick heart has been studied using the radiolabeled antagonist [³H]quinuclidinyl benzilate (QNB). In assays containing only buffer and a source of receptor protein, the antagonist radioligand bound to a single, high affinity state of the receptor. If Mg²⁺ and EDTA were added, [³H]QNB bound to a single, low affinity state. The guanine nucleotide analog, guanylylimidodiphosphate [Gpp(NH)p], reversed the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ so that [³H]QNB again bound only to a single, high affinity state. Sodium could also reverse the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ on antagonist binding but the effects of sodium and Gpp(NH)p on [³H]QNB binding were not additive.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1)

Studies of the localization of the Na⁺-dependent sugar transport in monolayers of LLC PK₁ cells show that the uptake of a methyl α-d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [³H]phlorizin binding to monolayers of LLC PK₁ cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na⁺-dependent process. Binding at the low affinity sites on the contrary is Na⁺-independent. The mode of action of Na⁺ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na⁺ dependence and the matching of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for high affinity binding sites with the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of phlorizin for the inhibition of methyl α-d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α-d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na⁺ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na⁺-dependent sugar transport system in the apical membrane of LLC PK₁ cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.     

Solubilization and anionic regulation of cerebral sedative/convulsant receptors labeled with [35S] tert-butylbicyclophosphorothionate (TBPS)

Binding activity for the cage convulsant [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate, which appears to label a site closely associated with the chloride ionophore of the GABA_A/benzodiazepine receptor complex has been solubilized from rat cerebral cortex using the zwitterionic detergent CHAPS. Of several detergents screened, only CHAPS and CHAPSO were capable of solubilizing the binding activity with good recovery. The pharmacologic specificity of soluble [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is very similar to the membrane state. In both the membrane and soluble state, [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is enhanced by anions which support inhibitory post-synaptic potentials (“Eccles anions”), suggesting that [³⁵S]-$\text{t}$-butylbicyclophosphorothionate may label chloride channels thought to be involved in these potentials. Since this solubilization procedure also preserves GABA and benzodiazepine binding and their regulation by drugs such as barbiturates, purification and isolation of the macromolecular complex including chloride channel and GABA-benzodiazepine sites may be feasible.     

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.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10^?8 M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10^?8 M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to $\text{3}\text{4}$ of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about $\text{3}\text{4}$ of that of untreated cytosol.     

Effects of the intracerebroventricular injection of antinociceptive doses of acetylcholine on the stereospecific binding of 3H-dihydromorphine

The antinociceptive effects of intraventricularly administered acetylcholine (ACh) and its congeners have been demonstrated by previous investigators. The opiate receptor binding concept was used in this study to investigate possible correlations between ACh antinociception and its effects on opiate stereospecific binding. ACh $\text{in vitro}$ decreased the stereospecific binding of ³H-dihydromorphine in mouse brain homogenates. Such decrease was also observed in the brain homogenates of mice which had been treated with ACh intracerebroventricularly (i.v.t.). The decrease in the stereospecific binding of ³H-dihydromorphine induced by (i.v.t.) acetylcholine was inhibited by naloxone, atropine, cyclazocine and pentazocine. The $\text{d}$-isomers of cyclazocine and pentazocine were more potent than the $\text{l}$-isomers in antagonizing the inhibitory effects of i.v.t. acetylcholine upon the stereospecific binding of ³H-dihydromorphine to mouse brain homogenates. The same stereospecificity of these two narcotic analgesics in blocking acetylcholine had been previously observed in the tail-flick test. It is suggested that the antinociceptive effects of acetylcholine are related to the inhibition of opiate stereospecific binding, the mechanism of which is yet to be understood.     

Prostaglandin E antagonist activity of 11,15-bisdeoxy prostaglandin E1 and congeners

$\text{d,?}$-11,15-bisdeoxy PGE₁ and certain of its congeners were shown to inhibit gerbil colon contractions induced by $\text{?}$-PGE₁. While some of these compounds were selectively antagonistic of PGE₁-induced contractions, others additionally inhibited the gerbil colon agonist activities of $\text{?}$-PGE_2α and acetylcholine. The PGE₁ inhibitory activity was apparently competitive in nature. With relatively weak potencies, the bisdeoxy PGE congeners displaced ³H-PGE₁ from a fat cell binding site, suggesting competition for a common, putative receptor. Structure-activity relationships and potential utility of these analogs are discussed.     

Solubilization of rat kidney benzodiazepine binding sites

The high-affinity binding site for [³H]Ro 5–4864 has been solubilized from rat kidney using 1% Triton X-100. After lowering the concentration of detergent and using a poly(ethylene glycol) γ-globulin assay, it has been possible to demonstrate solubilization of about 90% of the binding sites. A single soluble class of binding sites with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 1.8 nM is found. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Gel filtration revealed a major peak of binding activity with apparent molecular weight of 215000 and a Stokes' radius of 5.03 nm.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.     

Detection of nicotinic cholinergic transmission in malapteruruselectricus electrop lax

Biochemical and electrophysiological studies were conducted on the electric organ of the electric fish of the Nile, $\text{Malapterurus}$$\text{electricus}$, in order to determine if transmission was chemically mediated. There was no binding of [³H] acetylcholine, [³H] quinuclidinyl benzilate or [³H]-perhydrohistrionicotoxin; but low acetylcholinesterase activity was observed, as was binding of [¹²⁵I] α-bungarotoxin. The latter binding was detectable at 0.85 ± 0.07 pmol/g tissue, and was totally inhibited by 1 μM α-bungarotoxin or 100 μM d-tubocurarine. A tetrodotoxin-sensitive action potential was measured which was Na⁺- dependent. Depolarization (30–40 mV) was caused by carbamylcholine, and this was blocked by d-tubocurarine or α-bungarotoxin. The data suggest that this electric organ which may be a rich source for electrically excitable channels, is innervated by nicotonic cholinergic motoneurons, but the concentrations of acetylcholine receptors and acetylcholinesterase are very low.     

Carbamylcholine-induced rapid cation efflux from reconstituted membrane vesicles containing purified acetylcholine receptor.

Membrane preparations containing essentially only the four polypeptides considered to constitute the acetylcholine receptor are purified from $\text{Torpedo}\text{californica}$ electroplax. Treatment of these membranes with 2% ($\text{w}\text{v}$ aqueous sodium cholate followed by removal of all insoluble matter results in a solubilized purified receptor preparation that can be reassociated with phospholipids during dialysis to remove the detergent. Such reconstituted receptor is shown to retain the capability of translocating ²²Na⁺ across the membrane in response to carbamylcholine binding in a highly reproducible manner. The dose response for this effect is similar to that observed for the original electroplax membrane preparation and the carbamylcholine induced signal is completely blocked by α-bungarotoxin.     

Eosin,a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

(1) Eosin bound to the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of K⁺ has practically the same fluorescence as eosin without enzyme while in the presence of Na⁺ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K⁺ concentration but with a low affinity. With equal concentrations of Na⁺ and K⁺ more is bound in the presence of Na⁺, and the difference between 150 mM Na⁺ and 150 mM K⁺ shows one high-affinity eosin binding site per ³²P-labelling site ($\text{K}{}_{\mathrm{D}}$ 0.45 μM). With lower concentrations of the cations there are between one and two Na⁺-dependent high-affinity eosin binding sites per ³²P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na⁺-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na⁺ (+Mg²⁺). (4) Eosin, like ATP, increases the Na⁺ relative to K⁺ affinity $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{= 150}\text{mM}$) for Na⁺ activation of hydrolysis and for Na⁺ protection against inactivation by $\text{N-}\text{ethylmaleimide}$. (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na⁺ opens a high-affinity site for ATP while K⁺ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na⁺ relative to K⁺ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$ the system reacts with two different ATP molecules.     

Extraction of peripheral proteins from nicotinic acetylcholine receptor-enriched membranes

The solubilisation of membrane proteins from nicotinic acetylcholine receptor-enriched membranes from the electric organ of Torpedo marmorata was studied. Chaotropic ions were shown to be ineffective in extracting peripheral proteins from these membranes. Two different anhydrides, 2,3-dimethylmaleic and 3,4,5,6-tetrahydrophthalic anhydride, released certain peripheral membrane proteins but not the integral receptor protein. Treatment of membranes containing $\text{> 3}\text{nmol}$ α-bungarotoxin binding sites per mg protein with anhydride resulted in a 43 kDa polypeptide as the major constituent of the solubilised material. The nature of the 43 kDa polypeptide is discussed. Gentle anhydride treatment did not change the α-bungarotoxin and carbamoylcholine binding properties of the receptor.     

Affinity-labeling of purified acetylcholine receptor from Torpedo californica

The receptor for acetylcholine purified from electric tissue of $\text{Torpedo californica}$ has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.     

Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ + K+, and 5'-adenylylimidodiphosphate

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate. (AMP-P(NH)P. This compound, in which the oxygen connecting the β and γ phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg²⁺-ATPase activities. The $\text{K}{}_{\mathrm{<mtext>1</mtext>}}$ of AMP-P(NH)P for Mg²⁺-ATPase activity was 2.0 · 10^?4 M and, while the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of ATP for this activity was also 2.0 · 10^?4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na⁺, K⁺ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 · 10^?3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-fre porcine erythrocyte ghosts were studied in order to characterize the system more adequately.     

Rapid cation flux from Torpedo californica membrane vesicles: comparison of acetylcholine receptor enriched and selectively extracted preparations.

Rapid efflux of ²²Na from within closed vesicles derived from $\text{Torpedo}\text{californica}$ electroplax membranes has been studied as an $\text{in}\text{vitro}$ assay of acetylcholine receptor functionality. The most highly purified membrane preparations contained major polypeptides of M.W. 43 and 90 × 10³ daltons in addition to the four peptides characteristic of the acetylcholine receptor (40, 50, 60, 65 × 10³ daltons). Removal of these extra peptides by base extraction did not significantly alter the characteristics of carbamylcholine induced ²²Na efflux: the agonist dose response curve was similar, preequilibration with agonist caused desensitization, the irreversible antagonist α-Bungarotoxin blocked the efflux and the reversible blockade by the neurotoxin perhydrohistrionicotoxin was also retained. The dose response curve for perhydrohistrionicotoxin corresponded closely to its known binding characteristics for base extracted membranes.