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1.
The anterio-posterior distribution of cholinergic receptor binding sites in human hippocampus (five parts) as well as the effect of age (age range 3 days - 85 years) on receptor properties has been studied. Muscarinic binding sites was measured using labelled quinuclidinyl benzilate (3H-QNB) as ligand and labelled tubocurarine (3H-TC) was used for measurement of nicotine-like binding sites.The highest number of 3H-QNB binding sites in human hippocampus was measured at 3 days and 3 weeks of age and the lowest at 82 years of age. The proportion of high and low affinity muscarinic binding sites respectively was about the same at all ages investigated.A decrease in 3H-QNB binding sites with age was found in the anterior parts of the hippocampus (age range 55–84 years). When individual data for number of 3H-TC binding sites were plotted against corresponding number of 3H-QNB binding sites a strong correlation was observed in most of the different regions of the hippocampus.  相似文献   

2.
A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED50 values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both 3H-GABA and 3H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of 3H-GABA and 3H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of 3H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of 3H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of 3H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of 3H-GABA and 3H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.  相似文献   

3.
The characteristics of the Na+-independent high-affinity binding of [3H]GABA to various types of crude synaptic membranes (CSM) prepared from rat brain cortex were studied. In freshly prepared CSM the content of GABA was so high that the high-affinity [3H]GABA binding could not be determined. In contrast when the frozen-thawed CSM were incubated at 37° for 30 min with or without Triton X-100 or phospholipase C and then washed repeatedly, there was a virtual disappearance of GABA from the supernatant extracts and the binding constants of [3H]GABA to CSM could be determined. Two apparent populations of [3H]GABA binding sites, one with a low- and the other with a high-affinity constant, were detected. The ratio of the number of high- to low-affinity binding sites varies with the method used to prepare the membranes. The lowest value of this ratio was observed with membranes incubated at 37° for 30 min. However, when frozen-thawed CSM were treated with 0.05% Triton X-100 repeatedly, the ratio of the number of high- to low-affinity binding sites increased progressively. This increase in ratio is due to a selective increase in the number of the high-affinity sites without significant changes in the number of the low-affinity sites. The extent of the increase in the number of sites that bind [3H]GABA with high affinity after repeated Triton X-100 treatments was paralleled by a decrease of an endogenous protein which inhibits GABA binding. The reapplication of this endogenous material to membranes repeatedly treated with Triton X-100 reduces the number of high-affinity binding sites for [3H]GABA to values similar to those measured in membranes that were not treated with Triton X-100. The inhibitory preparation extracted from CSM incubated with Triton X-100 was shown to be free of GABA or phospholipids. The gel filtration chromatography reveals the presence of two molecular forms of the inhibitor; of these, the high-molecular-weight material fails to bind GABA, whereas the low-molecular-weight material appears to bind GABA. The high-molecular-weight endogenous inhibitor has been termed GABA modulin.  相似文献   

4.
Abstract: The binding of [3H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [3H]γ-aminobutyric acid ([3H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN?. An apparent single class of binding sites for [3H]BMC (KD= 30 nM) was observed in 0.1 M SCN? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The Bmax was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [3H]GABA. Frozen membranes, however, showed decreased levels of [3H]BMC binding with no decrease or an actual increase in [3H]GABA binding sites. [3H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [3H]GABA binding. Kinetics experiments with [3H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [3H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [3H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [3H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [3H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.  相似文献   

5.
[3H]Flunitrazepam (FNZ) binding to cortical neurons from fetal rat brain was investigated in vitro. The use of a synthetic medium specific for neurons made it possible to plot a developmental curve of3H-FNZ binding in an almost pure neuronal culture. Detectable specific binding was present in vitro at time 0 (that is, the 16th gestational day). A progressive increase of binding, due to an increment in the number of recognition sites, was observed on the subsequent days. The affinity of the specific binding sites to3H-FNZ was enhanced by the addition of exogenous GABA, whereas the density was not affected.  相似文献   

6.
Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [3H]GA4 binding sites (Kd ~ 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the Kd for [3H]GA4 increased, whereas the maximum number of saturable [3H]GA4 binding sites did not change significantly. The dissociation of [3H]GA4 from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [3H]GA4 dissociation from its binding sites. These results implied that AC-94,377 and [3H]GA4 compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.  相似文献   

7.
Specific and saturable binding of 125I-bovine albumin to rat adipocytes in suspension was observed (apparent Kd 2.09 ± 0.52 × 10?6 M; 8.58 ± 2.49 × 106 sites per cell; mean ± SEM). The binding was rapid and reversible for at least 10 min, suggesting that endocytosis of albumin was minor under assay conditions. Pre-incubation of cells with epinephrine bitartrate caused an apparent increase in number and decrease in affinity of the adipocyte binding sites for albumin. These findings suggest that a specific and saturable interaction of albumin with the adipocyte surface may play a role in the cellular uptake and release of free fatty acids.  相似文献   

8.
The regional distribution and in vivo binding of the dopamine analog 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN) was studied in the brain. The highest density of binding sites was in the striatum, with virtually no binding in the cerebellum. The binding of [3H]ADTN reflects an occupation of specific dopamine sites because the binding was diminished by the simultaneous administration of the dopamine antagonist haloperidol or the dopamine precursorl-3,4-dihydroxyphenylalanine (l-dopa). Chronic administration of haloperidol orl-dopa prior to assaying for in vivo binding resulted in an increase in the number of sites for [3H]ADTN which correlates to the increase observed in in vitro assays following long-term treatment with these agents. The subcellular distribution of in vivo labeled ADTN sites in the caudate nucleus indicate a high density of specific binding sites in the microsomal fraction, P3. Overall, these data demonstrate that the aminotetralins, such as ADTN, which bind with high affinity to the dopamine receptor in the caudate nucleus in vitro and in vivo, can provide precise information on the topography of this receptor.  相似文献   

9.
Saturable binding sites for tritiated dihydromorphine ([3H]DHM), D-Ala2-D-Leu5-enkephalin ([3H]DADL) and etorphine were found in a crude synaptosomal preparation of bovine retina. Scatchard analysis of saturation binding curves of each ligand was curvilinear and the presence of two independent binding sites inferred. The density of binding sites of [3H]etorphine was similar to that reported in brain crude synaptosomal preparations, and the affinity for the high affinity binding site to each ligand was similar to values determined in brain. Moreover, the regulation of the binding sites by GTP and sodium was also similar to that observed in brain. Selective binding sites for [3H]DADL (δ-sites) were not detectable, although binding sites similar in nature to μ-binding sites were detected.  相似文献   

10.
Recent studies have demonstrated the presence of high affinity binding sites for [3H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [3H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [3H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [3H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [3H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [3H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [3]] imipramine selectively labels serotonin uptake sites in brain and platelet.  相似文献   

11.
Maier T  Yu C  Küllertz G  Clemens S 《Planta》2003,218(2):300-308
Metal-binding domains consisting of short, contiguous stretches of amino acids are found in many proteins mediating the transport, buffering, trafficking or detoxification of metal ions. Phytochelatin synthases are metal-activated enzymes that function in the detoxification of Cd2+ and other toxic metal and metalloid ions. In order to localize Cd2+-binding sites, peptide libraries of two diverse phytochelatin synthases were synthesized and incubated with 109Cd2+. Distinct binding sites and binding motifs could be localized based on the patterns of Cd2+-binding. The number of binding sites was consistent with previous findings for recombinant protein. Positions of binding sites appeared to be conserved even among diverse phytochelatin synthases. Mutant peptide analysis was used to assess the contribution of exemplary amino acids to binding. Several binding motifs contain cysteines or glutamates. For cysteines a strong correlation was found between binding activity and degree of conservation among known phytochelatin synthases. These findings indicate the suitability of peptide scanning for the identification of metal-binding sites. The functional role of several cysteines was investigated by expression of hemagglutinin-tagged phytochelatin synthases in phytochelatin synthase-deficient, Cd2+-hypersensitive Schizosaccharomyces pombe cells. The data are consistent with a model suggesting functionally essential metal-binding activation sites in the N-terminal catalytic part of phytochelatin synthases and additional binding sites at the C-terminus not essential for activity.Abbreviations EMM Edinburgh's minimal medium - GSH glutathione - HA hemagglutinin - PC phytochelatin - PCS phytochelatin synthase  相似文献   

12.
[3H]-Ouabain binding to muscle preparations was utilized to estimate the number of Na+,K+-ATPase enzyme units in hindlimbs from 8 week old lean and obese mice. Specific [3H]-ouabain binding per mg particulate protein was 36% lower in obese mice; whereas, the affinity of the binding sites for ouabain was similar in obese and lean mice. Since obese mice had less muscle than lean mice, the number of Na+,K+-ATPase enzyme units in hindlimbs from obese mice was less than half the number observed in lean mice.  相似文献   

13.
《Life sciences》1987,40(15):1537-1543
The pineal gland and particularly its major hormone, melatonin, may participate in several physiological functions, including sleep promotion, anticonvulsant activity and the modulation of biological rhythms and affective disorders. These effects may be related to an interaction with benzodiazepine receptors, which have been demonstrated to be present in the pineal gland of several species including man. The present study examined the characteristics of benzodiazepine binding site subtypes in the human pineal gland, using [3H] flunitrazepam and [3H] PK 11195 as specific ligands for central and peripheral type benzodiazepine binding sites respectively. Scatchard analysis of [3H] flunitrazepam binding to pineal membrane preparations was linear, indicating the presence of a single population of sites. Clonazepam and RO 15-1788, which have a high affinity for central benzodiazepine binding sites, were potent competitors for [3H] flunitrazepam binding in the human pineal, whereas RO 5-4864 had a low affinity for these sites. Analyses of [3H] PK 11195 binding to pineal membranes also revealed the presence of a single population of sites. RO 5-4864, a specific ligand for peripheral benzodiazepine binding sites was the most potent of the drugs tested in displacing [3H] PK 11195, whereas clonazepam and RO 15-1788 were weak inhibitors of [3H] PK 11195 binding to pineal membranes. Overall, these results demonstrate, for the first time, the coexistence of peripheral and central benzodiazepine binding sites in the human pineal gland.  相似文献   

14.
Opiate receptor-ligand binding was investigated in brains of young and aged female F344 rats. A significant reduction in the density of binding sites for 3H-dihydromorphine was observed in the thalamus and midbrain of aged rats. Evidence of two binding sites was observed in the anterior cortex of young rats, whereas aged rats exhibited only a single site. The occurrence of pituitary tumors in the old female rats did not affect 3H-dihydromorphine binding. The highest density of dihydromorphine binding sites was found in the diencephalon, and the lowest density in the hippocampus. Receptor densities in the anterior cortex, striatum, amygdala, frontal poles and midbrain were intermediate, and few, if any, high affinity binding sites for dihydromorphine were found in the pons-medulla or posterior cortex.  相似文献   

15.
The binding of (±)-[3H]isoproterenol and (—)-[3H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a Kd of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a Kd of 195 nM and has 2200 sites/cell. The binding of (—)-[3H]dihydroalprenolol shows one type of site with a Kd of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[3H]isoproterenol as compared to the (—)-[3H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.  相似文献   

16.
Preliminary studies indicate the presence of PGF specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 × 10?9M and the concentration of binding sites of ~0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF specific binding sites indicates specificity for the 9α-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific 3H-PGE1 binding could be demonstrated, no high affinity sites could be quantitated. 3H-PGE1 binding appeared unaffected by changes in temperature or time of incubation, whereas PGF specific binding was significantly modified by both these factors.  相似文献   

17.
Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [3H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [3H]-dopamine binding indicates an IC50 for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [3H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems.  相似文献   

18.
Abstract : The influence of β‐amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (‐73 to ‐87%) as well as in sporadic Alzheimer's disease (AD) cases (‐37 to ‐57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both α3 and α4 and to α4 nAChR subtypes, respectively. Saturation binding studies with [3epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (‐82%) for the high‐affinity site was observed in APP 670/671 subjects with no change in KD compared with controls (0.018 nM APP 670/671 ; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.  相似文献   

19.
[3H]Prostaglandin (PG) E2 bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE2 and PGE1 competed for the [3H]PGE2 binding site. The PGs inhibited in the following decreasing order: PGE2 = PGE1 ? PGF > PGA2 > PGF > PGB2. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 ± 0.14 × 10?9. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10?11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10?11 mol binding sites/g. The results suggest that there is a high-affinity PGE2 receptor present in both plasma membrane and sarcoplasmic reticulum.  相似文献   

20.
Abstract

Autoradiograms of rat brain sections were compared obtained from animals receiving a tritiated drug through intravenous injection or from precut sections incubated in vitro. The benzomorphan analogue 3H-(?)-bremazocine was used as ligand and its distribution to all different opioid binding sites was followed. Although the general distribution of opioid binding sites visualized on 3H-LKB ultrofilms was independent of the methodological approach used, the maximal number of such sites (Bmax) was greater in brain sections incubated in vitro than after in vivo drug application. Since the number of binding sites is highly dependent on the particular incubation condition used, this finding has no further relevance.  相似文献   

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