Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.)

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5′-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3′,5′-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5′-deiodinase activity measured at rT3 concentrations up to 10 μM and in the absence of DTT does not saturate appreciably. In the presence of 1 mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5′-deiodination in liver is potently stimulated by 1 mM DTT. The marked biochemical differences between 5′-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts.     

Selective binding of imatinib to the genetic variants of human α1-acid glycoprotein

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to α₁-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of K_a = 1.7(± 0.2) × 10⁶ M^− 1 and n = 0.94 could be determined for the binding on the F1-S variant at 37°. Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nK_a ≈ 3 × 10⁴ M^− 1) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.     

Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.     

High-mannose N-glycan-specific lectin from the red alga Kappaphycus striatum (Carrageenophyte)

From a fresh sample (1 kg) of cultivated red alga Kappaphycus striatum, three isolectins, KSA-1 (15.1 mg), KSA-2 (58.0 mg) and KSA-3 (6.9 mg), were isolated by a combination of extraction with aqueous ethanol, ethanol precipitation, and ion exchange chromatography. Isolated KSAs were monomeric proteins of about 28 kDa having identical 20 N-terminal amino acid sequences to each other. Their hemagglutination activities were not inhibited by monosaccharides, but inhibited by glycoproteins bearing high-mannose N-glycans. In a binding experiment with pyridylaminated oligosaccharides by centrifugal ultrafiltration-HPLC assay, the isolectin KSA-2 was exclusively bound to high-mannose type N-glycans, but not to other glycans. Including complex types and a pentasaccharide core of N-glycans, indicating that it recognized branched oligomannosides. The binding activity of KSA-2 was slightly different among high-mannose N-glycans examined, indicating that the lectin has a higher affinity for those having the exposed (α1-3) Man in the D2 arm. On the other hand, KSA-2 did not bind to a free oligomannose that is a constituent of the branched oligomannosides, implying that the portion of the core GlcNAc residue(s) of the N-glycans is also essential for binding. Thus, KSA-2 appears to recognize the extended carbohydrate structure with a minimal length of a tetrasaccharide, Man(α1-3)Man(α1-6)Man(β1-4)GlcNAc. This study indicates that K. striatum, which has extensively been cultivated as a source of carrageenan, is a good source of a valuable lectin(s) that is strictly specific for high-mannose N-glycans.     

alpha3-galactosylated glycoproteins can bind to the hepatic asialoglycoprotein receptor.

In mammals, clearance of desialylated serum glycoproteins to the liver is mediated by a galactose-specific hepatic lectin, the 'asialoglycoprotein receptor'. In humans, serum glycoprotein glycans are usually capped with sialic acid, which protects these proteins against hepatic uptake. However, in most other species, an additional noncharged terminal element with the structure Galalpha1-->3Galbeta1-->4R is present on glycoprotein glycans. To investigate if alpha3-galactosylated glycoproteins, just like desialylated glycoproteins, could be cleared by the hepatic lectin, the affinities of alpha3-galactosylated compounds towards this lectin were determined using an in vitro inhibition assay, and were compared with those of the parent compounds terminating in Galbeta1-->4R. Diantennary, triantennary and tetraantennary oligosaccharides that form part of N-glycans were alpha3-galactosylated to completion by use of recombinant bovine alpha3-galactosyltransferase. Similarly, desialylated alpha1-acid glycoprotein (orosomucoid) was alpha3-galactosylated in vitro. The alpha3-galactosylation of a branched, Galbeta1-->4-terminated oligosaccharide lowered its affinity for the membrane-bound lectin on whole rat hepatocytes 50-250-fold, and for the detergent-solubilized hepatic lectin 7-50-fold. In contrast, alpha3-galactosylation of asialo-alpha1-acid glycoprotein caused only a minor decrease in affinity, increasing the IC50 from 5 to 15 nM. Fully alpha3-galactosylated alpha1-acid glycoprotein, intravenously injected into the mouse, was rapidly cleared from the circulation, with a clearance rate close to that of asialo-alpha1-acid glycoprotein (t1/2 of 0.42 min vs. 0.95 min). Its uptake was efficiently inhibited by pre-injection of an excess asialo-fetuin. Organ distribution analysis showed that the injected alpha1-acid glycoprotein accumulated predominantly in the liver. Taken together, these observations suggest that serum glycoproteins that are heavily alpha3-galactosylated will be rapidly cleared from the bloodstream via the hepatic lectin. It is suggested that glycosyltransferase expression in murine hepatocytes is tightly regulated in order to prevent undesired uptake of hepatocyte-derived, circulating glycoproteins.     

Inhibitory effect of glycoprotein isolated from Opuntia ficus-indica var. saboten MAKINO on activities of allergy-mediators in compound 48/80-stimulated mast cells

The present study was performed to investigate the anti-allergy potentials of glycoprotein (90 kDa) isolated from Opuntia ficus-indica var. saboten MAKINO (OFI glycoprotein) in vivo (ICR mice) and in vitro (RBL-2H3 cells). At first, to know whether the OFI glycoprotein has an inhibitory ability for allergy in vivo, we evaluated the activities of allergy-related factors such as histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in compound 48/80 (8 ml/kg BW)-treated ICR mice. After that, we studied to found the effect for anti-allergy in vitro such as nuclear factor kappa B (NF-κB) and inducible nitric oxide synthase (iNOS), extracellular signal-regulated kinase (ERK) 1/2, arachidonic acid, and cyclooxygenase-2 (COX-2) in compound 48/80 (5 μg/ml)-treated RBL-2H3 cells. Our results showed that the OFI glycoprotein (5 mg/kg) inhibited histamine and β-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in mice serum. Also OFI glycoprotein (25 μg/ml) has suppressive effects on the expression of MAPK (ERK1/2), and on protein expression of anti-allergic proteins (iNOS and COX-2). Thus, we speculate that the OFI glycoprotein is an example of natural compound that blocks anti-allergic signal transduction pathways.     

High affinity sialoside ligands of myelin associated glycoprotein

Myelin associated glycoprotein (Siglec-4) is a myelin adhesion receptor, that is, well established for its role as an inhibitor of axonal outgrowth in nerve injury, mediated by binding to sialic acid containing ligands on the axonal membrane. Because disruption of myelin-ligand interactions promotes axon outgrowth, we have sought to develop potent ligand based inhibitors using natural ligands as scaffolds. Although natural ligands of MAG are glycolipids terminating in the sequence NeuAcα2-3Galβ1-3(±NeuAcα2−6)GalNAcβ-R, we previously established that synthetic O-linked glycoprotein glycans with the same sequence α-linked to Thr exhibited ∼1000-fold increased affinity (∼1 μM). Attempts to increase potency by introducing a benzoylamide substituent at C-9 of the α2-3 sialic acid afforded only a two-fold increase, instead of increases of >100-fold observed for other sialoside ligands of MAG. Surprisingly, however, introduction of a 9-N-fluoro-benzoyl substituent on the α2-6 sialic acid increased affinity 80-fold, resulting in a potent inhibitor with a K_d of 15 nM. Docking this ligand to a model of MAG based on known crystal structures of other siglecs suggests that the Thr positions the glycan such that aryl substitution of the α2-3 sialic acid produces a steric clash with the GalNAc, while attaching an aryl substituent to the other sialic acid positions the substituent near a hydrophobic pocket that accounts to the increase in affinity.     

Colorectal cancer metastases affect the biochemical characteristics of the human liver β-adrenoceptor-G-protein-adenylate cyclase system

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked β₂-adrenoceptors (β₂-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of β₂-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of β-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of β-ARs (B_max) was significantly reduced, compared to NA-NM liver tissues (40.09 ± 2.83 vs. 23.09 ± 3.24 fmol/mg protein; P < 0.001). A similar decrease in the β-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6 ± 2.2 vs. 23.8 ± 3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized β-ARs were uncoupled from stimulatory G-protein (G_S), as total density of β-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition–concentration curves of ICI 118.551 showed the presence of a homogeneous population of the β₂-AR subtypes. Neither the binding patterns nor the inhibition constant (K_i) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic β-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence β-AR-mediated functions in both organs.     

Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and Iow cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

Functional characterization of the Escherichia coli Fis-DNA binding sequence

The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis-DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis-DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis-DNA binding affinity. The results revealed that: (1) the − 7G and + 3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C⁵ methyl group of thymine, when present at the + 4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis-DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from − 6 to − 4, from − 2 to + 2, and from + 4 to + 6), with only moderate penalties on the binding affinity, the roughly similar contributions of − 3A or G and + 3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.     

Isolation of human platelet glycoproteins

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of M_r ≈ 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of M_r ≈ 165 000.Treatment of whole platelets by periodate oxidation and sodium[³H]borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of M_r ≈ 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with M_r ≈ 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others.Phosphorylation of intact platelets with ³²P_i also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the     

Screening the bioactive compounds in aqueous extract of Coptidis rhizoma which specifically bind to rabbit lung tissues β2-adrenoceptor using an affinity chromatographic selection method

A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to β₂-adrenoceptor (β₂-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a β₂-AR affinity column. The retention compounds eluted from the β₂-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the β₂-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized β₂-AR. Their association constants were (2.28 ± 0.11) × 10⁴/M and (3.00 ± 0.10) × 10⁴/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized β₂-AR, and the corresponding association constants were (2.20 ± 0.09) × 10⁻⁴/M and (6.78 ± 0.001) × 10⁵/M.     

Detoxification of sulfur mustard by enzyme-catalyzed oxidation using chloroperoxidase

One of the most interesting methods for the detoxification of sulfur mustard is enzyme-catalyzed oxidation. This study examined the oxidative destruction of a sulfur mustard by the enzyme chloroperoxidase (EC 1.11.1.10). Chloroperoxidase (CPO) belongs to a group of enzymes that catalyze the oxidation of various organic compounds by peroxide in the presence of a halide ion. The enzymatic oxidation reaction is affected by several factors: pH, presence or absence of chloride ion, temperature, the concentrations of hydrogen peroxide and enzyme and aqueous solubility of the substrate. The optimum reaction conditions were determined by analyzing the effects of all factors, and the following conditions were selected: solvent, Britton–Robinson buffer (pH = 3) with tert-butanol (70:30 v/v); CPO concentration, 16 U/mL; hydrogen peroxide concentration, 40 mmol/L; sodium chloride concentration, 20 mmol/L. Under these reaction conditions, the rate constant for the reaction is 0.006 s⁻¹. The Michaelis constant, a measure of the affinity of an enzyme for a particular substrate, is 1.87 × 10⁻³ M for this system. The Michaelis constant for enzymes with a high affinity for their substrate is in the range of 10⁻⁵ to 10⁻⁴ M, so this value indicates that CPO does not have a very high affinity for sulfur mustard.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Cholecalciferol (vitamin D 3) improves cognitive dysfunction and reduces inflammation in a rat fatty liver model of metabolic syndrome

Aim

The aim of this study was to examine the effects of cholecalciferol on systemic inflammation and memory in the setting of fatty liver disease in rats.

Materials and methods

To induce the development of fatty liver disease, the rats were fed a 35% fructose solution over 8 weeks. Group I (n = 6) was designated as the control group and fed with standard rat chow. Group II (n = 6) was provided with, standard rat chow, and 0.3 μg/kg/day of oral cholecalciferol over a duration of 2 weeks. In addition to standard rat chow, group III (n = 6) and group IV (n = 6) were given 4 mL of the 35% fructose solution per day via oral gavage for 8 weeks. However, group IV was also given 0.3 μg/kg/day of oral cholecalciferol over 2 weeks. After the treatment period, passive avoidance tasks were performed by all groups. The liver and brain were harvested for subsequent biochemical and histopathologic analyses.

Key findings

The development of fatty liver extends the memory latency period of passively avoiding tasks after 1 trial. Moreover, there were increases in brain TNF-α and plasma MDA levels according to two-way analysis of variance. Cholecalciferol supplementation decreased the latency period of passively avoiding tasks in rats with hepatosteatosis, and also significantly reduced brain TNF-α and plasma MDA levels.

Significance

Fatty liver may contribute to the development of systemic inflammation, which affects cognition and causes deficits in memory; however, the anti-inflammatory and antioxidant properties of vitamin D may improve the cognitive function of rats with hepatosteatosis.     

Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid

As part of ongoing work aimed at generating proteolytically stable, readily applicable, radiolabeled endomorphin-2 (EM-2) analogs for elucidation of the topological requirements of peptide binding to μ-opioid receptors, we report here on the synthesis, radiolabeling, binding kinetics and binding site distribution of an EM-2 analog in which Pro² is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. [³H][(1S,2R)ACHC]²EM-2 (specific activity 63.49 Ci × mmol⁻¹) bound specifically to its binding sites with high affinity (K_D = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, B_max of 151 ± 4 fmol × mg protein⁻¹ in rat brain membranes. A similar affinity value was obtained in kinetic assays. Both Na⁺ and Gpp(NH)p decreased the affinity, proving the agonist character of the radioligand. Specific μ-opioid ligands displaced the radioligand with much higher affinities than did δ- and κ-ligands. The autoradiographic distribution of the binding sites of [³H][(1S,2R)ACHC]²EM-2 agreed well with the known locations of the μ-opioid receptors in the rat brain. In consequence of its high affinity, selectivity and enzymatic resistance [19], the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding.