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1.
A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the alpha-, beta- and delta-chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, alpha 248, beta 254, and delta 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position delta 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.  相似文献   

2.
T Heidmann  J P Changeux 《Biochemistry》1986,25(20):6109-6113
The kinetics of covalent labeling of the alpha, beta, gamma, and delta chains of the acetylcholine receptor (AcChR) from Torpedo marmorata by the noncompetitive blocker [3H]chlorpromazine ([3H]CPZ) are investigated by using rapid mixing photolabeling techniques. In an initial study [Heidmann, T., & Changeux, J. P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1897-1901], it was shown that the rate of [3H]CPZ labeling increases 100-1000-fold upon simultaneous addition of nicotinic agonists to the AcChR and that prior addition of these agonists abolishes the effect. The data were interpreted in terms of the rapid labeling of the transient active state of the AcChR where the ion channel is in its open configuration. This interpretation was recently challenged [Cox, R. N., Kaldany, R. R. J., Di Paola, M., & Karlin, A. (1985) J. Biol. Chem. 260, 7186-7193] on the ground of studies with a different noncompetitive blocker, [3H]quinacrine azide, and the suggestion was made that this compound labels the rapidly desensitized closed channel conformation of the AcChR. In this paper it is shown that the rate of rapid labeling of the AcChR by [3H]CPZ decreases to negligible values upon exposure of the AcChR to nicotinic agonists, in the 100-500-ms time range. The absolute values of the rate constants of this decrease (10-15 s-1 for saturating concentrations of acetylcholine and carbamoylcholine) and their variation with agonist concentration (apparent dissociation constants of 40 microM and 0.4 mM for acetylcholine and carbamoylcholine, respectively) are those expected for the rapid desensitization of the AcChR.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
P Muhn  A Fahr  F Hucho 《Biochemistry》1984,23(12):2725-2730
Photoaffinity labeling of the nicotinic acetylcholine receptor from Torpedo marmorata electric tissue was performed in the presence of cholinergic effectors in the millisecond to second time range by a combination of a stopped-flow apparatus and a high-energy pulse laser. The label applied was [3H]triphenylmethylphosphonium, a lipophilic cation previously shown to be a specific blocker of the acetylcholine receptor ion channel. With the receptor in the resting state most of the label was incorporated into the alpha polypeptide chains. In the presence of agonists and antagonists increasing incorporation into the delta- and (less pronounced) the beta-chain was observed. The time course of this increase had a half-life of about 0.4 s, being slower than receptor activation and channel opening. in the resting, active, and even rapidly desensitized state, the alpha polypeptide chains appear to be the primary targets of the photoaffinity reaction. The action spectrum of the photolabeling has a sharp maximum at lambda = 270 nm and a small-side maximum at lambda = 290 nm. It does not resemble the absorption spectrum of the label and may hint at amino acid side chains as the moieties activated by UV light causing the photolabeling. The effector specificity of the observed slow increase of label incorporation into the delta polypeptide chain was investigated. It does not prove that slow desensitization is the underlying event. The agonists acetylcholine and carbamoylcholine as well as treatment of receptor-rich membranes with phospholipase A2 (but not phospholipase D) triggered labeling of delta, but antagonists such as D-tubocurarine and most conspicuously flaxedil had a similar effect.  相似文献   

4.
We have studied alkylation of the acetylcholine receptor by N-[3H]ethylmaleimide ([3H]NEM) under various conditions. The radiolabeled preparations were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the receptor complex into subunits, and the incorporation of 3H into each type of chain was determined. We found the following: (i) When cysteines of native receptor in intact membranes were reacted with [3H]NEM, only the beta-subunit was labeled; the extent of alkylation did not change significantly if cholinergic effectors were present during this reaction. (ii) When the disulfide bonds of the receptor were reduced with dithiothreitol (DTT), the alpha- and beta-chains were labeled with [3H]NEM. The presence of receptor agonists and competitive antagonists during alkylation significantly altered the labeling patterns. Gallamine and hexamethonium markedly enhanced, while carbamylcholine and decamethonium markedly lessened, labeling of the alpha-subunit. Choline, d-tubocurarine, and alpha-neurotoxin induced small, but significant decreases in alkylation of the alpha-subunit, while procaine had no effect. (iii) When the same ligands were present during the reduction step, subsequent labeling with [3H]NEM produced patterns similar to those described in (ii). We also investigated the effects of gallamine and hexamethonium on reduction of the disulfide bond located near the acetylcholine binding site by using the affinity alkylating reagent (bromoacetyl)choline (BAC). Gallamine (0.1 mM) was able to increase the rate of reduction of this particular disulfide bond 3-fold in comparison to the control. In these experiments, alkylation by BAC blocked 50% of the toxin binding sites. Hexamethonium (1 mM) had a similar effect.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
The muscarinic acetylcholine receptor was solubilized, in a sensitive form for GTP and Na+, from bovine cerebral cortex using a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The solubilized muscarinic receptor displayed characteristics as follows: (1) high affinity to nanomolar concentration of Z-[3H]quinuclidinyl benzilate; (2) muscarinic agonists and antagonists had similar inhibitory potencies as on the membrane-bound receptor; (3) without Na+, GTP did not significantly alter the binding affinity of muscarinic agonists and antagonists; (4) GTP in the presence of Na+, selectively decreased the affinity of muscarinic agonists, carbamylcholine and oxotremoline, but not the antagonist binding affinity; (5) Na+ in the absence or presence of GTP, reduced both muscarinic agonist and antagonist affinities.  相似文献   

6.
Local anesthetics and other noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor, acting at sites other than the acetylcholine-binding sites, block channel opening and/or cation translation through the open channel. In order to characterize the NCI sites and to decide among possible mechanisms of NCI action, we have photolabeled the receptor in membrane from Torpedo electric tissue with the photolyzable NCI [3H]quinacrine azide ([3H]QA), using a continuous-flow, rapid-mixing device and millisecond-duration irradiation. Membrane, [3H]QA, and effectors were mixed, and, after delay times of 20 ms or greater, the mixture was irradiated for 2 ms, quenched, and collected. Brief exposure of the receptor to acetylcholine, but not to hexamethonium or d-tubocurarine, induced a state particularly susceptible to photoincorporation of [3H]QA. This acetylcholine-induced photoincorporation was exclusively into the alpha and beta chains of the receptor, peaked at 100-ms delay time, declined to 15% of maximum after delay times of minutes, and was blocked by the NCIs proadifen and histrionicotoxin. At 20-ms delay, the dependence of labeling by 2 microM [3H]QA on acetylcholine concentration was characterized by an apparent dissociation constant of about 15 microM and a Hill coefficient of 1. The kinetics of the development of susceptibility to photolabeling and the apparent lack of positive cooperativity in the effect of acetylcholine on this development suggest that the preferentially photolabeled state is a transient, rapidly developing, desensitized state, rather than an open-channel state.  相似文献   

7.
We have characterized the time-resolved labeling of a site on the Torpedo californica electrocyte acetylcholine receptor (ACHR) by the photoreactive noncompetitive inhibitor derivative quinacrine azide (QA). The dependence of [3H]QA labeling on acetylcholine (ACH) concentration and on time is consistent with the preferential labeling by [3H]QA of ACHR in the open state. The ACH-dependent [3H]QA labeling, which was associated predominantly with the alpha-subunit, was blocked by other noncompetitive inhibitors including quinacrine, chlorpromazine, proadifen, histrionicotoxin, and bupivacaine. alpha-Subunit from ACHR labeled with [3H]QA 20 ms after the addition of ACH was cleaved with CNBr, and the fragments were separated by high pressure liquid chromatography. A peptide containing a major site of specific labeling was purified on two different reverse-phase columns. By N-terminal sequencing, amino acid composition, binding to mercurial-agarose, and apparent molecular weight, this [3H]QA-labeled peptide was identified as alpha-208-243, a CNBr fragment containing the putative membrane-spanning helix M1.  相似文献   

8.
R E Middleton  J B Cohen 《Biochemistry》1991,30(28):6987-6997
The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.  相似文献   

9.
P Muhn  F Hucho 《Biochemistry》1983,22(2):421-425
The lipophilic cation [3H]triphenylmethylphosphonium, frequently used as a voltage sensor in membrane systems, binds reversibly to a site different from the acetylcholine binding site. This is concluded from the different pH dependences of the binding of these two ligands. Furthermore [3H]triphenylmethylphosphonium, previously identified as a channel blocker, can be covalently incorporated into acetylcholine receptor-rich membranes from Torpedo electric tissue by UV irradiation of the receptor-ligand complex. In the absence of effector, predominantly the alpha-polypeptide chains (Mr 40000) of the receptor protein are labeled by the radioactive ligand. The agonist carbamoylcholine strongly stimulates the labeling, but it directs the label predominantly to the delta- and beta-polypeptide chains. The antagonist D-tubocurarine and the virtually irreversible competitive antagonist alpha-bungarotoxin have qualitatively the same effect as the agonist carbamoylcholine. Significant differences were obtained with receptor-rich membranes prepared from Torpedo marmorata and Torpedo californica: No agonist- or antagonist-stimulated reaction was observed with the latter. The results are interpreted as an indication of a rearrangement of the receptor's quaternary structure caused by cholinergic effector binding preceding discrimination between agonists and antagonists.  相似文献   

10.
Previous reports from this laboratory have demonstrated that alpha 2-adrenergic receptors accelerate Na+/H+ exchange in NG108-15 neuroblastoma X glioma cells and evoke platelet secretion via a pathway involving Na+/H+ exchange. The present studies were designed to examine whether agents that interact with Na+/H+ antiporters also might influence alpha 2-adrenergic receptor-ligand interactions. We observed that Na+ decreases receptor affinity for the agonists epinephrine, norepinephrine, and UK14304 and slightly increases receptor affinity for the antagonists yohimbine and idazoxan in digitonin-solubilized preparations from porcine brain cortex. Increases in [H+] also decrease receptor affinity for agonists and cause either a slight increase or no change in receptor affinity for antagonists. Amiloride analogs accelerate the rate of [3H] yohimbine dissociation from digitonin-solubilized receptors with a relative effectiveness that parallels their ability to block Na+/H+ exchange in other systems. Interestingly, these modulatory effects of Na+,H+ and 5-amino-substituted analogs of amiloride are retained in homogeneous preparations of the alpha 2-adrenergic receptor, suggesting that the allosteric-binding sites for these agents are on the receptor-binding protein itself.  相似文献   

11.
We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.  相似文献   

12.
The binding of 3H-labeled 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) to isolated rat liver plasma membranes and its inhibition by PAF agonists and receptor antagonists was demonstrated. The specific binding was readily saturable with a high affinity. The equilibrium dissociation constant (KD) value was 0.51 (+/- 0.14) nM and the maximal number of binding sites (Bmax) was estimated to be 141 (+/- 18) fmol/mg protein. The binding site was PAF specific-biologically inactive enantiomer was practically inactive. Two PAF-like receptor antagonists, Ono-6240 and CV-3988, and two PAF-unlike receptor antagonists, L-652,731 and kadsurenone, also displaced the binding of [3H]PAF to rat liver plasma membranes but their relative potencies in this system differed from those found in other receptor systems. Mg2+ potentiated [3H]PAF binding but inhibited it at concentrations higher than 10 mM. Both Na+ and K+ inhibited the Mg2+-potentiated binding, an ionic effect which was different from that found in rabbit platelets. These results suggest that rat livers contain PAF-specific receptors, and the receptors in rat livers are different from those found in other receptor systems.  相似文献   

13.
Sustained activation of muscarinic acetylcholine receptors on neuron-like NG108-15 hybrid cells reduces the number of [3H]quinuclidinyl benzilate binding sites per cell as much as 88%. The response occurs at concentrations of agonists commensurate with those needed to occupy receptors and inhibit adenylate cyclase. Decreases in steady-state receptor levels persist as long as activator remains present. Withdrawal of activator results in a slow increase in receptor levels that is blocked by cycloheximide. Activation shortens receptor half-life by a factor of nearly 4, indicating that regulation occurs at the level of receptor breakdown.  相似文献   

14.
A comparison was made of the actions of phencyclidine receptor agonists and N-methyl-D-aspartate (NMDA) receptor antagonists in two well-defined neurochemical test systems. These included (i) [3H]acetylcholine release from striatal cholinergic interneurons in vitro, a system known to be positively modulated by corticostriatal excitatory amino acid inputs in vivo; and (ii) cerebellar cGMP levels in vivo, an indicator of cerebellar Purkinje cell activity, which is also modulated by excitatory amino acid inputs. Using these neuronal systems, we report that phencyclidine receptor agonists demonstrated a noncompetitive antagonism of NMDA receptor agonist actions.  相似文献   

15.
16.
To characterize the structure of the agonist-binding site of the Torpedo nicotinic acetylcholine receptor (AChR), we have used [3H]acetylcholine mustard [( 3H]AChM), a reactive analog of acetylcholine, to identify residues contributing to the cation-binding subsite. Reaction of [3H]AChM, in its aziridinium form, with AChR-rich membrane suspensions, resulted initially in reversible, high affinity binding (K approximately 0.3 microM) followed by slow alkylation of the acetylcholine-binding site. Incorporation of label into AChR alpha-subunit was inhibited by agonists and competitive antagonists, but not by noncompetitive antagonists, and reaction with 3 microM [3H]AChM for 2 h resulted in specific alkylation of 0.6% of alpha-subunits. Within the alpha-subunit, greater than 90% of specific incorporation was contained within an 18-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Val-46 and containing N-linked carbohydrate. To identify sites of specific alkylation, [3H]AChM-labeled alpha-subunit was digested with trypsin, and the digests were fractionated by reverse phase high pressure liquid chromatography. Specifically labeled material was recovered within a single peak containing a peptide extending from Leu-80 to Lys-107. NH2-terminal amino acid sequencing revealed specific release of 3H in cycle 14 corresponding to alpha-subunit Tyr-93. Identification of Tyr-93 as the site of alkylation was confirmed by radiosequence analysis utilizing o-phthalaldehyde to establish that the released 3H originated from a peptide containing prolines at residues 2 and 9. Because [3H]AChM contains as its reactive group a positively charged quaternary aziridinium, alpha-subunit Tyr-93 is identified as contributing to the cation-binding domain of the AChR agonist-binding site. The selective reaction of [3H]AChM with tyrosyl rather than acidic side chains indicates the importance of aromatic interactions for the binding of the quaternary ammonium group, and the lack of reaction with the tyrosyl or acidic side chains within alpha 190-200 emphasizes the selective orientation of acetylcholine within its binding site.  相似文献   

17.
The Torpedo nicotinic acetylcholine receptor (AChR) contains a binding site for aromatic amine noncompetitive antagonists that is distinct from the binding site for agonists and competitive antagonists. To characterize the location and function of this allosteric antagonist site, an alkylating analog of meproadifen has been synthesized, 2-(chloroethylmethylamino)-ethyl-2, 2-diphenylpentanoate HCl (meproadifen mustard). Reaction of [3H]meproadifen mustard with AChR-rich membrane suspensions resulted in specific incorporation of label predominantly into the AChR alpha-subunit with minor incorporation into the beta-subunit. Specific labeling required the presence of high concentration of agonist and was inhibited by reversible noncompetitive antagonists including proadifen, meproadifen, perhydrohistrionicotoxin (HTX), and tetracaine when present at concentrations consistent with the binding affinity of these compounds for the allosteric antagonist site. No specific alkylation of the AChR alpha-subunit was detected in the absence of agonist, or in the presence of the partial agonist phenyltrimethylammonium or the competitive antagonists, d-tubocurarine, gallamine triethiodide, or decamethonium. Reaction with 35 microM meproadifen mustard for 70 min in the presence of carbamylcholine produced no alteration in the concentration of [3H]ACh-binding sites, but decreased by 38 +/- 4% the number of allosteric antagonist sites as measured by [3H]HTX binding. This decrease was not observed when the alkylation reaction was blocked by the presence of HTX. These results lead us to conclude that meproadifen mustard alkylates the allosteric antagonist site in the Torpedo AChR and that part of that site is associated with the AChR alpha-subunit.  相似文献   

18.
Photoaffinity labeling of the beta-adrenergic receptor   总被引:3,自引:0,他引:3  
A new photoactive beta-adrenergic antagonist, p-azidobenzylcarazolol (pABC) has been synthesized by combining a carbazole moiety with a p-azido-benzyl substituent. The compound has been labeled with tritium to a specific activity of 26 Ci/mmol. In frog erythrocyte membranes, [3H]p-azido-benzylcarazolol binds to the beta-adrenergic receptor with the expected beta 2 specificity and with high affinity (KD congruent to 100 +/- 10 pM). Unlabeled p-azido-benzylcarazolol can irreversibly inactivate the [3H]dihydroalprenolol-binding activity of frog erythrocyte membranes in a photodependent manner which can be prevented by beta-adrenergic agents. Incubation of frog erythrocyte membranes or digitonin-solubilized preparations of these membranes or digitonin-solubilized preparations of these membranes which had been enriched in beta-adrenergic receptors by a Sepharose-alprenolol chromatography step led to covalent incorporation of radioactivity into a Mr = 58,000 peptide. Specific incorporation of [3H]pABC into the Mr = 58,000 peptide could be prevented by both beta-adrenergic agonists and antagonists. This peptide has previously been purified and shown to contain the beta-adrenergic receptor-binding site (Shorr, R. G. L., Lefkowitz, R. J., and Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826). Thus, photoaffinity labeling of the beta-adrenergic receptor protein directly identifies the same hormone-binding subunit as has been isolated by conventional purification techniques.  相似文献   

19.
The nature of the interactions between the N-methyl-D-aspartate (NMDA) and the phencyclidine (PCP) receptors was studied in membranes obtained from rat cerebral cortex and washed repeatedly to remove endogenous excitatory amino acids. Binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to its receptor sites in these membranes proceeded slowly and did not reach equilibrium even after incubation for 4 h at 25 degrees C. The dissociation rate of [3H]TCP-receptor complexes was also slow (t1/2 = 128-165 min). Both association and dissociation followed first-order reaction kinetics, with similar time constants (0.0054 min-1). Addition of glutamate and glycine to the washed membranes was immediately followed by a marked increase in the rates of both association of [3H]TCP with the receptors and its dissociation from them (t1/2 = 8 min). Association now followed second-order reaction kinetics. Accelerated association of [3H]TCP with its binding sites could also be induced by NMDA or by glutamate alone, and glycine enhanced the effect. All effects of glutamate and glycine on [3H]TCP binding kinetics were blocked by the competitive NMDA receptor antagonist AP-5 [D-(-)-2-amino-5-phosphovaleric acid]. [3H]TCP-receptor interactions at equilibrium were not altered by AP-5 or by glutamate and glycine. The binding data were fitted to a model in which interactions of [3H]TCP with the receptor involve a two-step process: the outside ligand must cross a barrier (presumably a closed NMDA receptor channel in the absence of agonists). Once agonists are added, this limitation is removed (presumably because the channel is open).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.  相似文献   

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