Binding of a tritiated enkephalinergic analog (FK 33-824) to a mitochondrial fraction of rat brain

FK-33-824 (Try-D-Ala-Gly-MePhe-Met(O)ol) is a potent enkephalin analog which has been tritium labelled with a high specific radioactivity (41 Ci/mmole). The labelled drug exhibits specific and saturable binding to rat brain crude mitochondrial fraction. Specific binding is inhibited by low concentrations of morphine, levallorphan and beta-endorphin, suggesting that FK 33-824 [3H] binds preferentially to mu opiate sites. Binding studies at equilibrium and kinetics of formation and dissociation of the labelled ligand-receptor complex indicate that FK 33-824 [3H] binds to two classes of specific sites. Their affinities are distinguishable at 0 degree (KD = 1.3 and 5.8 nM) and very close to each other at 37 degree (KD = 1.9 nM).     

Binding of [3H]DMCM, a Convulsive Benzodiazepine Ligand, to Rat Brain Membranes: Preliminary Studies

DMCM (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) produces convulsions in mice and rats, probably by interacting with benzodiazepine (BZ) receptors. Investigation of specific binding of [3H]DMCM to rat hippocampus and cortex revealed polyphasic saturation curves, indicating a high-affinity site (KD = 0.5-0.8 nM) and a site with lower affinity (KD = 3-6 nM). BZ receptor ligands of various chemical classes, but not other agents, displace [3H]DMCM from specific binding sites--indicating that [3H]DMCM binds to BZ receptors in rat brain. The regional distribution of [3H]DMCM binding is complementary to that of the BZ1-selective radioligand [3H]PrCC. Specific binding of [3H]DMCM (0.1 nM) was reduced by gamma-aminobutyric acid (GABA) receptor agonist to approximately 20% of the control value at 37 degrees C in chloride-containing buffers; the reduction was bicuculline methiodide- and RU 5135-sensitive. The effective concentrations of 10 GABA analogues in reducing [3H]DMCM binding correlated closely to published values for their GABA receptor affinity. Specific binding of [3H]DMCM is regulated by unknown factors; e.g. enhanced binding was found by Ag+ treatment of membranes, in the presence of picrotoxinin, or by exposure to ultraviolet light in the presence of flunitrazepam. In conclusion, [3H]DMCM appears to bind to high-affinity brain BZ receptors, although the binding properties are different from those of [3H]flunitrazepam and [3H]PrCC. These differences might relate in part to subclass selectivity and in part to differences in efficacy of DMCM at BZ receptors.     

Binding of [3H]batrachotoxinin A-20-alpha-benzoate to a high affinity site associated with house fly head membranes

1. [3H]Batrachotoxinin A-20-alpha-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly (Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands. 2. Specific binding of [3H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion. 3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom stimulated the specific binding of [3H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [3H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant (KD) for [3H]BTX-B of 80 nM and a maximal binding capacity (Bmax) of 1.5 pmol/mg protein. 4. Parallel experiments in the absence of venom gave a KD value of 140 nM and a Bmax of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [3H]BTX-B binding by increasing the affinity of this site approximately two-fold. 5. The specific binding of [3H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [3H]BTX-B to mammalian brain preparations did not affect the binding of [3H]BTX-B to house fly head membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine H1-receptor in the retina: species differences

Histamine H1-receptors in membranes of the various mammalian retinas were studied by [3H]mepyramine binding assay. Specific [3H]mepyramine bindings to bovine, pig, dog and human retinas were observed with the dissociation constants (KD), 3.8 +/- 1.2 nM, 1.8 +/- 0.6 nM, 2.6 +/- 0.6 nM and 3.0 +/- 0.9 nM, respectively, which were similar to those found in brains. But there was no detectable specific binding in the guinea-pig and rabbit retinas. The number of binding sites (Bmax) ranged from negligible value to 290.7 +/- 51.7 fmole/mg protein(human retina). Some H1-antagonists acted as potent agents in competing with [3H]mepyramine binding to bovine and pig retinas. These results indicated that histamine H1-receptors exist in some mammalian retina and have similar characteristics to those in brain membranes, but they distributes in the wide difference of the binding capacities among the species, while in brain variations were smaller.     

Ontogeny of Receptor Binding Sites for [3H]Glutamic Acid and [3H]Kainic Acid in the Rat Cerebellum

The development of the specific binding sites for L-[3H]glutamic acid (KD = 370 nM) and for [3H]kainic acid (KD = 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.     

The bindings of 3H-prazosin and 3H-yohimbine to alpha adrenoceptors in the guinea-pig stomach

Alpha adrenoceptor subtypes have been investigated by radioligand binding study in guinea-pig stomach using 3H-prazosin and 3H-yohimbine. The specific 3H-prazosin binding to guinea-pig stomach was saturable and of high affinity (KD = 1.4 nM) with a Bmax of 33 fmol/mg protein. Specific 3H-yohimbine binding to the tissue was also saturable and of high affinity (KD = 25.5 nM) with a Bmax of 150 fmol/mg protein. Adrenergic drugs competed for 3H-prazosin binding in order of prazosin greater than phentolamine greater than methoxamine greater than norepinephrine greater than clonidine greater than epinephrine greater than yohimbine. These drugs competed for 3H-yohimbine binding in order of yohimbine greater than phentolamine greater than clonidine greater than epinephrine greater than norepinephrine greater than prazosin greater than greater than prazosin greater than methoxamine. We also examined whether dopamine receptors exist in guinea-pig stomach, using radioligand binding study. Specific binding of 3H-spiperone, 3H-apomorphine, 3H-dopamine and 3H-domperidone was not detectable in the stomach. Dopaminergic drugs such as dopamine, haloperidol, domperidone and sulpiride competed for 3H-prazosin binding in order of haloperidol greater than domperidone greater than dopamine greater than sulpiride. Metoclopramide, sulpiride and dopamine competed for 3H-yohimbine binding in order of metoclopramide greater than sulpiride greater than dopamine. These results suggest that guinea-pig stomach has alpha 1 and alpha 2 adrenoceptors and has no specific dopamine receptors. It is also suggested that some dopamine receptor antagonists such as domperidone, haloperidol, sulpiride and metoclopramide have antagonistic actions on alpha adrenoceptors.     

Characterization of [3H]Mazindol Binding in Rat Brain: Sodium-Sensitive Binding Correlates with the Anorectic Potencies of Phenylethylamines

Saturable low-affinity binding sites for [3H]mazindol have been demonstrated in crude synaptosomal membranes from rat brain using both a centrifugation and a filtion assay. Studies on the regional distribution of these binding sites revealed that the hypothalamus and brainstem had the highest density of sites. Kinetic analysis of the binding of [3H]mazindol to hypothalamic membranes demonstrated a single class of noninteracting binding sites with an apparent affinity constant (KD) of 10.2 +/- 0.7 microM and maximal number of binding sites (Bmax) of 786 +/- 94 pmol/mg of protein. Specific [3H]mazindol binding was rapidly reversible, temperature sensitive, labile to pretreatment with proteolytic enzymes, and inhibited by physiological concentrations of sodium. In most peripheral tissues, such as the liver and kidney, very low levels of binding were observed; however, the adrenal gland had a relatively high density of sites. The potency of a series of anorectic drugs in inhibiting specific [3H]mazindol binding to hypothalamic membranes was highly correlated with their anorectic potencies in rats, but not with their motor stimulatory effects. These results suggest the presence of a specific drug recognition site in the hypothalamus that may mediate the anorectic activity of mazindol and related phenylethylamines.     

Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.     

Evidence for a non-opioid sigma binding site in the guinea-pig myenteric plexus

The presence of a binding site to (+)-(3H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site (KD = 46 +/- 5 nM; Bmax = 3.4 +/- 0.5 pmole/g wet weight) and a low affinity site (KD= = 342 +/- 72 nM; Bmax = 22 +/- 3 pmole/g wet weight). Morphine and naloxone 10(-4) M were unable to displace (+)-(3H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig.     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a KD of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu] enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H] GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their Ki values against [3H]GEMSA binding are similar to their Ki values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the Vmax for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the Bmax (maximal number of binding sites) for [3H]GEMSA is about 2,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H] GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the Ki of GEMSA against enzymatic hydrolysis and weakly inhibits [3H] GEMSA binding by increasing the KD.     

Characterization of the Binding of [3H]SR 95531, a GABAA Antagonist, to Rat Brain Membranes

A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.     

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.     

t-[3H]Butylbicycloorthobenzoate: New Radioligand Probe for the γ-Aminobutyric Acid–Regulated Chloride Ionophore

t-[3H]Butylbicycloorthobenzoate [( 3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25 degrees C, with 80-90% specific relative to total binding, a KD of 61 +/- 15 nM, and a Bmax of 1.6 +/- 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the gamma-aminobutyric acid receptor-ionophore complex.     

Binding and uptake of copper from ceruloplasmin

Specific binding of [67Cu]ceruloplasmin to plasma membrane containing preparations from rat tissues was shown in the presence of an excess of nonradioactive Cu(II) or ceruloplasmin. With Cu(II) there was positive cooperativity and an apparent KD of 10(-7) M. The effects of both "cold" ligands was partly additive. No "specific" binding was shown with Zn(II), unrelated proteins and after boiling the membranes. Total and specific binding of [67Cu]ceruloplasmin were 2-7 fold greater for heart and brain than for liver preparations, per g tissue or per mg protein, +/- correction for yield of 5'-nucleotidase. Cu(II) also inhibited uptake of [67Cu] from ceruloplasmin by CHO cells, but monensin did not, suggesting uptake of ceruloplasmin Cu occurs at the cell surface.     

Two distinct (-)nicotine binding sites in goldfish brain. Identification and characterization of putative neuronal nicotinic acetylcholine receptor subtypes

The binding of (-)-[3H]nicotine to membrane fragments and a detergent solubilized fraction of goldfish brain was characterized. (-)-[3H]nicotine binding was not displaced by alpha-bungarotoxin, but was displaced by (-)nicotine and carbamoylcholine with Ki of approximately 8.6 and 86 nM, respectively. Preincubation of solubilized membrane extract with alpha-bungarotoxin-coupled Sepharose resulted in the loss of approximately 50% of the (-)-[3H]nicotine binding protein from the eluent and an increase in (-)-[3H]nicotine binding to the gel compared to control, non-alpha-bungarotoxin Sepharose. 125I-alpha-bungarotoxin binding protein in the eluent from the same preincubation experiments was totally removed. In addition, incubation of the solubilized tissue extracts with alpha-bungarotoxin-coupled Sepharose resulted in an increase in the affinity for (-)-[3H]nicotine in the eluent (mean KD = 3.1) compared to control solubilized tissue extracts (KD = 6.4 nM). Specific (-)-[3H]nicotine binding sites could be eluted from the alpha-bungarotoxin-coupled Sepharose with carbamoylcholine and D-tubocurarine. Similar to previously reported 125I-alpha-bungarotoxin binding data, eye removal resulted in an approximately 40% decrease in (-)-[3H]nicotine binding in the contralateral tectum compared to that in the ipsilateral tectum. These data indicate that at least two distinct subtypes of (-)nicotine binding sites may be present in goldfish brain, one which binds alpha-bungarotoxin and (-)nicotine and another which binds only (-)nicotine.     

Specific binding of the calcium antagonist [3H]verapamil to membrane fractions from plants

Specific binding of the Ca2+ channel blocker [3H] verapamil to a membrane fraction from plants has been characterized. Binding to zucchini membranes was saturable and reversible. The apparent equilibrium dissociation constant is KD = 102 nM and the maximum number of binding sites is Bmax = 60 pmol/mg of protein. The KD determined from the association and dissociation rate constants is 130 nM. [3H]Verapamil binding to zucchini membranes could not be inhibited by the Ca2+ antagonists nifedipine and diltiazem. However, [3H]verapamil could be displaced by diltiazem but not by nifedipine from corn membranes. Sucrose density fractionation of zucchini membrane preparations revealed that [3H]verapamil binding sites are located primarily at the plasma membrane.