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PubMed1990

The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90.

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.

Affinity Labels↗
PubMed1994

Comparable 30-kDa apamin binding polypeptides may fulfill equivalent roles within putative subtypes of small conductance Ca(2+)-activated K+ channels.

Apamin, a peptide neurotoxin from bee venom, blocks small conductance Ca(2+)-activated K+ channels in central synapses and peripheral tissues. Using 125I-apamin, single classes of high affinity binding sites (Kd 1-3 pM) were identified on plasma membranes from rat, rabbit, guinea pig, and bovine brain and from rabbit, guinea pig, and bovine liver. Binding was sensitive to scyllatoxin, dequalinium, gallamine, and d-tubocurarine but not to charybdotoxin, toxin I, or mast cell degranulating peptide. In contrast, saturable binding of 125I-apamin to rat liver plasma membranes was virtually undetectable, thereby providing a correlation with the ability to measure apamin-sensitive Ca(2+)-activated potassium currents in rabbit and guinea pig hepatocytes but not in rat hepatocytes. In agreement with membrane binding studies, homobifunctional cross-linkers identified apparently identical 33-kDa 125I-apamin binding polypeptides on brain plasma membranes from all species and analogous but distinct polypeptides on plasma membranes from rabbit, guinea pig, and bovine liver. None of these affinity-labeled polypeptides were detectable on plasma membranes from rat liver. Affinity labeling was abolished on both liver and brain membranes by apamin, scyllatoxin, dequalinium, gallamine, and d-tubocurarine. These results indicate that comparable approximately 30-kDa polypeptides may fulfill equivalent functional roles within putative subtypes of apamin-sensitive small conductance Ca(2+)-activated K+ channels.

Animals↗
PubMed1996

Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: evidence for intracellular complexes.

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37 degrees C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Fig receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4 degrees C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4 degrees C and then washed, were shifted to 37 degrees C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex.

Affinity Labels↗
PubMed1985

Subunit structure of a high-affinity receptor for type beta-transforming growth factor. Evidence for a disulfide-linked glycosylated receptor complex.

The structure of high-affinity receptors for type beta-transforming growth factor (beta TGF) has been examined by affinity labeling with 125I-beta TGF and disuccinimidyl suberate. The major receptor component labeled by 125I-beta TGF in mouse, rat, and chick fibroblasts migrated as a 280-290-kilodalton species on dodecyl sulfate-polyacrylamide electrophoresis gels in the presence of reductant, dithiothreitol. A larger (330-kilodalton) species was labeled in human fibroblasts, but comparative peptide mapping indicated a close structural relationship with receptors from mouse fibroblasts. In the absence of reductant, the affinity-labeled beta TGF receptor migrated in the gels as a larger disulfide-linked complex. The molecular mass calculated from the hydrodynamic properties of native nonreduced beta TGF receptors was 565 (mouse) or 615 kilodaltons (human). Other molecular parameters for the beta TGF receptor were: Stokes radius, 8.3-8.5 nm; sedimentation coefficient, 12.7-13.0 S; and frictional ratio, f/f0 = 1.4. The beta TGF receptor was solubilized under conditions in which the structural and ligand-binding properties of the native state were retained. beta TGF receptors solubilized from human, mouse, and chick cells interacted specifically with immobilized wheat germ agglutinin. These data suggest that the high affinity receptor for beta TGF in human, rodent, and avian fibroblasts is a disulfide-linked glycosylated 565-615-kilodalton complex with a 280-330-kilodalton subunit that contains the ligand-binding site. The oligomeric structure of the beta TGF receptor does not appear to be induced by receptor occupancy with the ligand.

Affinity Labels↗
PubMed2002

Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.

Proteomics research requires methods to characterize the expression and function of proteins in complex mixtures. Toward this end, chemical probes that incorporate known affinity labeling agents have facilitated the activity-based profiling of certain enzyme families. To accelerate the discovery of proteomics probes for enzyme classes lacking cognate affinity labels, we describe here a combinatorial strategy. Members of a probe library bearing a sulfonate ester chemotype were screened against complex proteomes for activity-dependent protein reactivity, resulting in the labeling of at least six mechanistically distinct enzyme classes. Surprisingly, none of these enzymes represented targets of previously described proteomics probes. The sulfonate library was used to identify an omega-class glutathione S-transferase whose activity was upregulated in invasive human breast cancer lines. These results indicate that activity-based probes compatible with whole-proteome analysis can be developed for numerous enzyme classes and applied to identify enzymes associated with discrete pathological states.

Affinity Labels↗
PubMed1989

Multimeric structure of the tumor necrosis factor receptor of HeLa cells.

The tumor necrosis factor (TNF) receptor of HeLa cells was solubilized in Triton X-100 and characterized by gel filtration, affinity labeling, and ligand blotting studies. Receptors solubilized with Triton X-100 eluted in gel filtration as a major peak of Mr = 330,000 and retained high affinity binding (KD = 0.25 nM). Affinity labeling of soluble receptor/125I-TNF complexes using the reversible, bifunctional bis[2-(succinimidooxycarbonyl-oxy)ethyl] sulfone resulted in the formation of cross-linked species of Mr = 310,000, 150,000-175,000, 95,000, and 75,000. The formation of these complexes was competitively inhibited by unlabeled TNF. Partial reversal of cross-linking in these complexes and their analysis by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 125I-TNF dimers cleaved from the 95,000 band and 125I-TNF monomer cleaved from the 75,000 band, providing evidence for a Mr approximately 60,000 subunit. In addition, the 95,000 and 75,000 bands were resolved as components of larger complexes (Mr = 150,000-175,000), which presumably contain two receptor subunits. The Mr 95,000 and 75,000 bands were also released from the Mr 310,000 complex by reduction with dithiothreitol, suggesting a role for disulfide bond stabilization. To investigate the association of the putative receptor subunits, Triton X-100 extracts from HeLa membranes were fractionated by SDS-PAGE without reduction and transferred electrophoretically to nylon membranes for TNF binding assays. Only two bands of Mr = 60,000 and 70,000 specifically bound TNF, and higher Mr binding activity was not observed. These results indicate that TNF receptors in HeLa cells are high molecular weight complexes containing Mr = 60,000 and 70,000 subunits each capable of binding TNF and that the complexes are primarily stabilized by non-covalent, hydrophobic interactions.

Affinity Labels↗
PubMed2004

The structure-function relationship in the clostripain family of peptidases.

In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain. The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time-dependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme-BDD complex is KD = 74.6 +/- 2.1 micro m and the maximal rate constant of inactivation is k3 = 0.21 x min(-1). Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester (BAEE). Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site-directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition-derived alignment with caspases. A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position. Two possible binding sites for the calcium required for auto-activation could be located. Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.

Affinity Labels↗
PubMed2005

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity.

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.

Carbon Isotopes↗
PubMed1991

Use of photoaffinity probes containing poly(ethylene glycol) spacers for topographical mapping of the cholecystokinin receptor complex.

To further define the structure of the pancreatic cholecystokinin (CCK) receptor and the topographical distance relationships between its subunits, we developed a series of monofunctional photoaffinity probes in which a fixed receptor-binding domain was separated from a photolabile nitrophenylacetamido group by defined lengths of a flexible spacer. The well-characterized CCK receptor radioligand 125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33] provided the receptor-binding component of the probes, while the polymer poly(ethylene glycol) (2, 4, 7, and 10 monomer units long) was used as the spacer. The patterns of affinity labeling of rat pancreatic plasma membranes were examined as a function of spacer length. This ranged from 7.3 to 16.2 A, as calculated by root-mean-square end-to-end distances and validated experimentally by time-resolved fluorescence resonance energy transfer measurements. All probes in the series specifically labeled the Mr = 85,000-95,000 glycoprotein with Mr = 42,000 core, which has been proposed to contain the hormone recognition site. In addition, when the spacer length reached 16.2 A, membrane proteins of Mr = 80,000 and Mr = 40,000 were specifically labeled. The product of endo-beta-N-acetylglucosaminidase F digestion of the Mr = 80,000 protein was Mr = 65,000, similar to a protein previously identified in affinity labeling experiments using a CCK-33-based probe. These observations are consistent with the Mr = 85,000-95,000 pancreatic protein representing the hormone-binding subunit of the CCK receptor, while proteins of Mr = 80,000 and Mr = 40,000 may represent noncovalently associated subunits sited within 16.2 A of the binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosaminidase↗
PubMed1989

[Catalytic properties of acetylcholinesterase, modified by N,N-dimethyl-2-phenylaziridinium. An alkane sulfonation reaction].

By means of affinity labelling with N,N-dimethyl-2-phenylaziridinium ion (DPA) two forms of acetylcholinesterase were synthesized that contained one or two molecules of the label covalently attached to the enzyme. The reaction of native and covalently modified acetylcholinesterases with n-alkane sulfonyl chlorides CnH2n + 1SO2Cl at n = 1 -4 was used to characterize the reactivity and properties of the enzymes. It was found that labelling of acetylcholinesterase with one molecule of DPA did not affect the enzyme's reactivity. Acetylcholinesterase containing two labels (the second one presumably located at the anionic centre of the enzyme) displayed enhanced and more specific reactivity towards alkane sulfonyl chlorides. It was found that the phenomenon of acceleration caused by affinity modification is analogous to the influence of n-tetraalkylammonium ions on the same reaction. Therefore, the mechanism of regulation of the properties of the esteric centre, caused by affinity labelling of the enzyme at the anionic centre, is the same as in the case of n-tetralkylammonium ions.

Acetylcholinesterase↗
PubMed1979

Photoaffinity labeling of rat liver ribosomes by N-(2-nitro-4-azidobenzoyl)puromycin.

N-(2-Nitro-4-azidobenzoyl)-[3H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes.

Affinity Labels↗
PubMedDate not supplied

[Comparison of ATP-pyrophosphate exchange reactions, catalyzed by two different "one-site" forms of tryptophanyl-tRNA-synthetase and by native enzyme].

Kinetic parameters of the reaction of ATP-PPi exchange, catalysed by "one-site" form of beef pancreas tryptophanyl-tRNA-synthetase are determined. The one-site form was obtained by affinity labelling of one active centre of the enzyme with alkylated analog of tryptophan. The comparison of kinetic parameters of this one-site form with the other one produced earlier by affinity labelling with analog of tryptophanyl-tRNA and the native enzyme was carried out. It was shown that the presence of tRNA improves the discrimination properties of the enzyme and diminishes the concentrations of "dead-end" complexes. Negative cooperativity in binding of two molecules of tryptophan is observed, which disappears in the presence of ATP, leading to an apparent synergistic effect. The molecule of tryptophan, which is present in the active centre, diminishes affinity of pyrophosphate.

Adenosine Triphosphate↗
PubMed1990

Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils.

We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.

Binding, Competitive↗
PubMed1986

Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit.

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.

Acetylglucosaminidase↗
PubMedDate not supplied

[Affinity modification of Escherichia coli ribosomes by 4-[(N-2-chloroethyl)N-methylamino]benzyl-5'-phosphamide hexauridylate in a complex stabilized by codon-anticodon interactions on P and A sites].

Affinity labeling of E. coli ribosomes with 4-[(N-2-chloroethyl)-N-methylamino] benzyl-5'-phosphamide of hexauridylate was studied within the complex containing tRNAPhe at P site and Phe-tRNAPhe at A site directed by EF-Tu and GTP. Ribosomal proteins as well as rRNA both in 30S and 50S subunits were found to be labelled within the complex. Labeled proteins were identified as S3, S9 and L2. Selectivity of affinity labeling with mRNA analogs was shown to depend on the functional state of the ribosomes. Modification was more selective within the complex stabilized by codon-anticodon interaction both at A and P-sites than within the complex in which this interaction takes place preferentially at P site.

Anticodon↗
PubMed1995

Identification of a human type II receptor for bone morphogenetic protein-4 that forms differential heteromeric complexes with bone morphogenetic protein type I receptors.

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-beta-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. We have isolated a cDNA encoding a novel transmembrane serine/threonine kinase from human skin fibroblasts which we demonstrate here to be a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. Furthermore, a subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection only of BRK-2 + BRK-3, suggesting that the different heteromeric BMP receptor complexes have different signaling potential.

Base Sequence↗
PubMed1984

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

Adenosine Triphosphate↗
PubMed1982

[3H]-guanfacine: a radioligand that selectively labels high affinity alpha2-adrenoceptor sites in homogenates of rat brain.

[3H]-guanfacine (N-amidino-2-(2,6-dichloro 3[3H] phenyl) acetamide hydrochloride; 24.2 Ci/mmol) has been used as a radioligand in homogenates of rat cerebral cortex. Specific binding of [3H]-guanfacine was linear with respect to tissue concentration (2.5-15 mg/ml), saturable and not markedly affected in the pH range 6.5-8.0. Analysis of the saturation of [3H]-guanfacine binding using an iterative least squares fitting procedure gave best fits to a single site model. [3H]-guanfacine binding was of high affinity (Kd 1.77 +/- 0.24 nM; n = 8) to a population of non interacting sites (nH 0.99 +/- 0.02; n = 8) with a density of 118.2 +/- 8.4 fmol/mg protein (n = 8). Highest levels of binding were achieved in cerebral cortex followed by thalamus greater than hypothalamus greater than medulla/pons greater than spinal cord greater than striatum greater than cerebellum. Binding was stereoselective with regard to the (-)-isomer of noradrenaline and the order of potency for displacement of [3H]-guanfacine by agonists was naphazoline greater than clonidine greater than (-)-adrenaline greater than (-)-alpha methylnoradrenaline greater than (-)-noradrenaline greater than (+/-)-alpha-methylnoradrenaline greater than (+)-noradrenaline greater than methoxamine greater than (+)-adrenaline greater than phenylephrine and by antagonists was phentolamine greater than dihydroergocryptine greater than piperoxane greater than yohimbine greater than prazosin greater than labetalol greater than indoramin suggested binding to alpha 2-adrenoceptors. The monovalent cations Na+ and K+ and also guanosine 5'-triphosphate (GTP) produced concentration-dependent inhibition whereas the divalent cations Ca2+, Mg2+, and Mn2+ first enhanced, then inhibited [3H]-guanfacine binding. Na+ (150 mM) or GTP (100 microM) produced marked reductions and Mn2+ (5 mM) marked increases in the number of receptor sites labelled by [3H]-guanfacine. 9 It is concluded that [3H]-guanfacine preferentially labels a high affinity state of the alpha 2- adrenoceptor in homogenates of rat cerebral cortex.

Adrenergic alpha-Agonists↗