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PubMed1990

Competitive antibody binding inhibition ELISA for the detection of Plasmodium falciparum antigen.

A competitive antibody binding inhibition ELISA to detect Plasmodium falciparum-infected cells in clinical specimens was developed. Optimum conditions developed included: 12.5 micrograms/ml of P. falciparum antigen for plate coating, 25 micrograms/ml of polyclonal rabbit anti-P. falciparum IgG, 30 minute incubation of a mixture of infected red blood cell extract with anti-P. falciparum IgG, dilution of 1:500 of alkaline phosphatase-conjugated anti-rabbit IgG, and reading of the absorbance values 60 min after adding the p-nitrophenyl phosphate substrate. Reproducibility of the assay against cultured P. falciparum-infected red blood cells varied according to parasitemia, the higher the parasitemia, the better the reproducibility. The sensitivity of the assay was approximately 110 parasites/10(6) red blood cells. The assay was applied to field conditions involving 103 cases with falciparum malaria, 38 cases with vivax malaria and 30 healthy controls. With the 10% antibody binding inhibition as a cutoff, 87.4% of falciparum cases and 26.3% of vivax cases were positive. After treatment, the majority of cases became parasitologically negative with the corresponding negative assay. Regression analysis showed only weak but statistically significant correlation between the percent inhibition with parasitemia (r = 0.38, p less than 0.001), and this was more clearly shown in patients with high parasitemia.

Animals↗
PubMedDate not supplied

Suitability of LLC-PK1 pig kidney cells for the study of drug action on renal cell cholesterol uptake: identification and characterization of low-density lipoprotein receptors.

INTRODUCTION: The purpose of this study was to identify and characterize the presence of low-density lipoprotein receptors (LDLr) in LLC-PK(1) cells. METHODS: LLC-PK(1) cells were assessed for the presence of LDLr by conducting dose-response, LDL specific binding and competitive studies with DiI-LDL, and Western blot and RT-polymerase chain reaction (PCR) analyses. Assay conditions with IgG-C7, a monoclonal antibody (mAb) to the LDLr, were optimized, including temperature, preincubation time, and concentration in LLC-PK(1) cells. RESULTS: LLC-PK(1) cells express LDL receptors as determined by LDL specific and competitive binding studies and Western blot and RT-PCR analysis (specific binding 0.5 ng DiI-LDL/mug of cellular protein). DISCUSSION: Taken together, these findings confirm the presence of LDL receptors on LLC-PK1 cells and support the appropriateness of using these cells in studies involving renal cell cholesterol uptake and metabolism.

Animals↗
PubMedDate not supplied

Competition for binding to multiple sites of human serum albumin for cholecystographic agents and sulfobromophthalein.

The binding of two cholecystographic agents, iophenoxate and iopanoate, to human serum albumin was studied with 11 putative competitors; the results were qualitatively consistent with competitive binding to common sites. A more precise analysis of competition was achieved with four pairs of compounds for which the free and bound concentration of each was determined. The results were analyzed by a computer program and the dissociation constants calculated for both binder and competitor at specified sites on albumin. With numbering based on the rank order of dissociation constants for iophenoxate, the highest binding of the four compounds occurs at different sites: iophenoxate at site I; iopanoate at site II, sulfobromophthalein at site III; and bromphenol blue at site II. For a given compound, there is close agreement in the calculated affinities at different sites regardless of the competitor.

Binding Sites↗
PubMed2005

Action of 24-epibrassinolide on a cell line of the beet armyworm, Spodoptera exigua.

The Spodoptera exigua cell line Se4 is sensitive for ecdysteroid activity stimulated by the insect molting hormone, 20-hydroxyecdysone (20E), showing a cease in cell proliferation (with 50% inhibition around 1 microM) and characteristic cell morphology changes with aggregation and formation of long filamentous cytoplasmic extensions. The bisacylhydrazine tebufenozide also triggered such typical cellular effects in Se4, and in addition, it showed an affinity for binding in competition with 3H-ponasterone A (PoA) that was similar to 20E (with 50% competition around 1 microM), confirming that such non-ecdysteroids display an ecdysteroid agonist activity. In contrast, when Se4 cells were incubated with the native plant hormone 24-epibrassinolide (24BR), none of the effects triggered by 20E were observed. Hence, a competition binding experiment with 3H-PoA demonstrated no affinity of 24BR for binding to the ecdysteroid receptor in the Se4 cell line. In another series of experiments, the Se4 cell line was tested in sensitivity response to increased acetylcholinesterase (AchE) activity after treatment with ecdysteroid active compounds. The AchE activity measured in the cell line is discussed in relation to inhibition by eserine. The obtained results suggest that 24BR exerted no ecdysteroid activity.

Acetylcholinesterase↗
PubMed1997

Upregulation of alpha1A- and alpha1B-adrenergic receptor mRNAs in the heart of cardiomyopathic hamsters.

An increased density of alpha1-adrenergic receptors (AR) has been linked to the development of necrotic lesions in the heart of hamsters with hereditary cardiomyopathy. To determine whether this increase results from an upregulation of the receptor mRNA(s), Northern blot analyses were carried out in the heart of 60-day-old control and cardiomyopathic hamsters using selective DNA probes for the three subtypes of alpha1-ARs (alpha1A, alpha1B and alpha1D). Transcripts for the three alpha1-ARs were detected in both control and cardiomyopathic hamsters. A two-fold increase in the alpha1A- and alpha1B-AR mRNA levels was observed in the cardiomyopathic hearts when compared to controls. In contrast, no change in the alpha1D-AR mRNA level could be detected. The enhancement in alpha1A- and alpha1B-AR mRNA levels was paralleled by a 20% increase in the total number of alpha1-ARs, as assessed by [3H]prazosin radioligand binding assays. Competition binding assays using subtype selective ligands indicated that the increased density of both alpha1A and alpha1B receptors contributes to the total alpha1-AR upregulation. Taken together, these data suggest that the early development of hereditary cardiomyopathy in hamsters is accompanied by a specific overexpression of the alpha1A- and alpha1B-ARs. A discrete increase of the alpha1-AR density could contribute to eliciting coronary microspasms, therefore participating in the development of focal necrotic lesions that are characteristic of the hamster cardiomyopathic model.

Animals↗
PubMed1986

Purification and binding site characterization of the circulating trophoblastic androgen-binding protein.

The trophoblastic androgen-binding protein (t-ABP) was purified 150-fold with a recovery of 51% from serum of patients with hydatidiform mole using various chromatographic techniques, successively affinity on concanavalin A, ion exchange on QAE-Sephadex A 50, gel filtration on Sephadex G 200 and chromatofocusing. The chromatofocusing step eliminated any trace of contaminating sex-hormone binding globulin. Competitive binding experiments using the purified material, [3H]dihydrotestosterone and various steroid derivatives allowed an attempt at characterizing the steroid-binding site of the protein. This latter possess respectively hydrophilic domains facing position 2 and 17 of the steroid molecule, a hydrophilic and proton donor sequence facing position 3 of the steroid molecule, hydrophobic regions facing positions 6, 11 and 16 of the steroid molecule and electron donor domains facing positions 1 and 6 of the steroid molecule. These characteristics are compared with those of the sex hormone-binding globulin (SHBG), rat epididymis androgen-binding protein (RABP) and rat prostate cytoplasmic androgen receptor (CAR) binding sites, respectively. The results of this specificity study indicate that the t-ABP behaves very similarly to CAR, although major differences are likely to exist between the binding sites of both proteins, particularly in the protein domains facing C-1 and C-2 of the steroid.

Androgen-Binding Protein↗
PubMed1987

Two subpopulations of alpha 1-adrenergic receptors with high and low affinity for agonists: short-term exposure to agonists reduced the high-affinity sites.

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including beta-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in alpha 1-adrenergic receptors induced by agonists. alpha 1-receptors were studied on DDT1 MF-2 smooth muscle cells (DDT1-MF-2 cells) by specific [3H]prazosin binding. In competition binding on membranes and on intact cells at 4 degrees C or at 37 degrees C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37 degrees C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [3H]prazosin binding inhibited by 20 microM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [3H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4 degrees C, but only 30% at 37 degrees C. On DDT1-MF-2 cells preequilibrated with [3H]prazosin at 4 degrees C, and then shifted to 37 degrees C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4 degrees C it is the native form of alpha 1-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37 degrees C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 microM norepinephrine. The nature of this low-affinity form was further investigated. On DDT1-MF-2 cells preincubated with the agonist and then extensively washed at 4 degrees C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [3H]prazosin at 4 degrees C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4 degrees C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37 degrees C may be due to the agonist-induced sequestration of alpha 1-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.

Animals↗
PubMed1987

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex.

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on [3H]flunitrazepam (FLU), [3H]muscimol (MUS), and [35S]t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with [3H]FLU and [3H]MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of [35S]TBPS at concentrations as high as 50 microM. Type II pyrethroids inhibited [35S]TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 microM. The data presented here suggest that the interaction of Type II pyrethroids with the gamma-aminobutyric acid (GABA) receptor-ionophore complex is restricted to a site near the TBPS/picrotoxinin binding site.

Animals↗
PubMed1979

Androgen, estrogen and progesterone receptors of the R3327H Copenhagen rat prostatic tumor.

Sucrose density gradient analysis of the R3327H tumor cytosol demonstrated the presence of both androgen and estrogen binding proteins. Competitive binding analysis with 17 beta-estradiol, 5 alpha-dihydrotestosterone, R1881, cyproterone acetate, and cortisol was consistent with the presence of 2 different binding sites for androgens and estrogens. Scatchard binding analysis was performed in the dorsal-lateral prostate as well as the R3327H tumor from normal Copenhagen rats. High affinity receptors for androgen and estrogen but not progesterone were found. However, in R3327H tumors relapsing following castration, the presence of high affinity receptors for progesterone were readily detectable.

Animals↗
PubMed1996

Ability of nitrobenzylthioinosine to cross the blood-brain barrier in rats.

Nucleoside transport inhibitors that cross the blood-brain barrier may be able to potentiate the neuroprotective effects of adenosine. We tested whether nitrobenzylthioinosine (NBMPR) crosses the blood-brain barrier in three types of experiments. First, intravenous injection of [3H]NBMPR and [14C]sucrose was performed. Brain volume of distribution and brain delivery were greater for [3H]NBMPR than for [14C]sucrose. Second, rats were injected intraperitoneally with NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR, or vehicle. Perchloric acid extracts of brains from rats treated with NBMPR-P inhibited [3H]NBMPR binding in competition binding assays nearly 3-fold more than extracts from brains of vehicle-treated animals. Third, cerebrospinal fluid (CSF) extracted from rats treated with NBMPR-P (10 mg/kg i.p.) contained 24.1 +/- 4.4 nM NBMPR while levels were undetectable in CSF from vehicle-treated rats. From these data, we conclude that NBMPR crosses the blood-brain barrier.

Animals↗
PubMed1997

Human vascular kinin receptors of the B2 type characterized by radioligand binding.

1. The human umbilical vein responds to bradykinin (BK) with contractions that are mediated by B2 receptors. In the present study, the corresponding vascular smooth muscle B2 binding sites have been investigated. 2. [3H]-BK, a full agonist labelled ligand, was used to demonstrate a single binding site giving a Kd value of 0.51+/-0.02 nM and a Bmax of 24+/-1 fmol mg(-1) protein. Scatchard plots were linear (r=0.98) in the 0.05-5 nM range of concentrations. Non-specific binding was found to be 30% of total binding. 3. Competition binding curves gave the following order of potency for various B2 receptor agonists: BK-[Hyp3]-BK > or = Lys-BK >> [Aib7]-BK >>> [desArg9]-BK, which is typical of B2 receptors. There was no binding to B1 receptors since the selective B1 receptor ligand, Lys-[desArg9]BK was inactive up to 10 microM (n=4). 4. Characterization of the binding site with antagonists, performed with three chemically distinct series of peptide and non-peptide compounds, revealed a high affinity of Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK) (Ki 0.17 nM; n=4) which was more potent that FR 173657 ([(E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinol inyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl] acrylamide]) (Ki 1.94 nM; n=4), D-Arg-[Hyp3,D-Phe7,Leu8]-BK (Ki 256 nM; n=4) and Win 64338 (phosphonium, [[4[[2[[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopro pyl]amino]phenyl]methyl]tributyl, chloride, monohydrochloride) (Ki 1,450 nM; n=4). 5. The present study describes and characterises B2 receptor binding sites in the vascular smooth muscle of the human umbilical vein. The binding assay appears to be suitable for studying new agonists or antagonists designed to activate or block the B2 receptor class that mediate the majority of the physiopathological effects of kinins in man.

Adult↗
PubMed1995

Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide.

The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

Amino Acid Sequence↗
PubMed1998

Up-regulation of the neuronal form of nitric oxide synthase in response to prolonged muscarinic M1 receptor stimulation.

It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M1 receptors and nNOS, with 3.3 microM or 1 mM carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by L-[3H]citrulline and cyclic [3H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [N-methyl-3H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M1 receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.

Animals↗
PubMed1985

Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites.

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin.

Animals↗
PubMed2006

Characterization of a radiolabeled small molecule targeting leukocyte function-associated antigen-1 expression in lymphoma and leukemia.

OBJECTIVE: Leukocyte function-associated antigen-1 (LFA-1) is constitutively expressed on leukocytes, including overexpression on lymphomas and leukemias. We have developed a derivative of BIRT 377, an allosteric inhibitor of LFA-1, which may be chemically tagged without affecting binding. In this study, we modified this derivative, (R)-1-(4-aminobutyl)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methylimidazolidine- 2,4-dione (butylamino-NorBIRT), and demonstrated its potential as a noninvasive imaging agent. METHODS: Specific binding of fluorescein-labeled butylamino-NorBIRT to both human and murine cells was demonstrated using equilibrium binding and dissociation techniques. A radiometal, lutetium-177 (Lu-177), was incorporated into the butylamino-NorBIRT through 1,4,7,10-tetraazacyclododecane-N,N',N",N'''- tetraacetic acid (DOTA) as a chelator. RESULTS: Equilibrium-binding experiments demonstrated that fluorescein- labeled butylamino-NorBIRT specifically binds human and murine LFA-1 with affinity constants of 135 and 186 nM, respectively. Dissociation kinetic experiments demonstrated an off-rate of 0.168/second(1) on murine cells, consistent with the observed affinity constant. Lutetium-177 was used for labeling, with > or =99.99% radiochemical purity and incorporation yield. This radiolabeled derivative exhibited high stability in fetal bovine serum (FBS) at 37 degrees C over 72 hours. (177)Lu-DOTA-butylamino-NorBIRT showed a binding affinity of 235 nM to human LFA-1 for equilibrium binding and competitive binding experiments. CONCLUSIONS: The radiolabeled DOTA-butylamino-NorBIRT may have potential as a noninvasive imaging or therapeutic agent in both human and mouse models.

Animals↗
PubMed1990

Synthesis of insulin-like growth factor I using N-methyl pyrrolidinone as the coupling solvent and trifluoromethane sulphonic acid cleavage from the resin.

Insulin-like growth factor I (IGF-I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N-methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double-coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S-acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF-I peptides were full agonists of natural IGF-I in a radioimmunoassay, in an IGF-I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF-binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF-I.

Animals↗
PubMed1976

Embryo cell surfaces--lectin binding and cell proliferation.

The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Concanavalin A decrease cell number and proportion of cells incorporating [3H] thymidine in case of 8- and 10-day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-day-old embryo cells. No effect was noticed in 12-day cells. These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8th to the 12th day of development, and slowly increases on the 16-day cells, the affinity constant remaining rather constant. The results indicate that the age-dependent effect of lectin on embryo cells could not be directly related to the number of lectin-binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of concanavalin A. Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.

Animals↗
PubMed2005

Binding profile of the endogenous novel heptapeptide Met-enkephalin-Gly-tyr in zebrafish and rat brain.

Zebrafish is considered a model organism, not only for the study of the biological functions of vertebrates but also as a tool to analyze the effects of some drugs or toxic agents. Five opioid precursor genes homologous to the mammalian opioid propeptide genes have recently been identified; one of these, the zebrafish proenkephalin, codes a novel heptapeptide, the Met-enkephalin-Gly-Tyr (MEGY). To analyze the pharmacological properties of this novel ligand, we have labeled it with tritium ([(3)H]MEGY). In addition, we have also synthesized two analogs: (d-Ala(2))-MEGY (Y-d-Ala-GFMGY) and (d-Ala(2), Val(5))-MEGY (Y-d-Ala-GFVGY). The binding profile of these three agents has been studied in zebrafish and rat brain membranes. [(3)H]MEGY presents one binding site in zebrafish, as well as in rat brain membranes, although it shows a slight higher affinity in zebrafish brain. The observed saturable binding is displaced by naloxone, thus confirming the opioid nature of the binding sites. Competition binding assays indicate that the methionine residue is essential for high-affinity binding of MEGY and probably of other peptidic agonists in zebrafish, whereas the change of a Gly for a d-Ala does not dramatically affect the ligand affinity. Our results show that the percentage of [(3)H]MEGY displaced by all the ligands studied is higher than 100%, thus inferring that naloxone (used to determine nonspecific binding) does not bind to all the sites labeled by [(3)H]MEGY. Therefore, we can deduct that some of the MEGY binding sites should not be considered classical opioid sites.

Amino Acid Substitution↗