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Biomedical subjects

J M Gershoni

Publications and source records attributed to J M Gershoni.

At least 19 recordsLinked to original sources
PubMed1991

Acetylcholine interactions with tryptophan-184 of the alpha-subunit of the nicotinic acetylcholine receptor revealed by transferred nuclear Overhauser effect.

Acetylcholine interactions with three genetically engineered fusion proteins containing peptides from the nicotinic acetylcholine receptor were studied by 1D and 2D nuclear magnetic resonance methods. The three proteins were Torpedo alpha 184-200, Torpedo alpha 186-198, and human alpha 183-204 of the acetylcholine receptor fused to the first 323 residues of the E. coli protein trpE. Nuclear Overhauser effect studies revealed interactions of bound acetylcholine with tryptophan-184 present in the Torpedo alpha 184-200, and the human alpha 183-204 sequences. These interactions are between the N(CH3)3+ and CH3 groups of acetylcholine with the aromatic protons of tryptophan. The appearance of these cross-peaks indicates a distance of less than 5 A between tryptophan and the bound ligand; however, direct contact has yet to be proven.

Acetylcholine↗
PubMed1991

Molecular dissection of cholinergic binding sites: how do snakes escape the effect of their own toxins?

Snakes have evolved a novel binding site demonstrating selective biorecognition. The snake nicotinic acetylcholine receptor is sensitive to acetylcholine while resistant to the effect of the lethal neurotoxins secreted in their own venom. By subjecting recombinant binding sites to point mutagenesis, biochemical analyses and NMR spectroscopy the binding characteristics of three cholinergic ligands have been measured. The amino acid residue at position 189 has been found to be of particular importance to toxin binding.

Amino Acid Sequence↗
PubMed1990

Comparison of the toxin binding sites of the nicotinic acetylcholine receptor from Drosophila to human.

Recombinant toxin binding proteins have been previously found to provide a convenient experimental system for the study of receptor-ligand recognition (Aronheim et al., 1988). Here, this system has been used to produce the binding sites of the cholinergic receptor derived from seven organisms, Torpedo californica, Xenopus, chick, mouse, calf, human, and Drosophila. These have been compared with respect to their toxin binding capacity. Scatchard analyses show that the KD values of alpha-bungarotoxin binding to the above sites are 63, 536, 150, 3200, 6200, 6470, and 1700 nM, respectively. These results reiterate the importance of alpha 183-204 as a ligand binding site. In order to increase the repertoire of sites available for study, chimeric structures were constructed. Through the analysis of such chimeras, some themes of the gross anatomy of the binding site can be learned. A positive subsite followed by a hydrophobic patch preceding a nucleophilic domain appears to be required for efficient toxin binding.

Animals↗
PubMed1990

Direct measurement of agonist binding to genetically engineered peptides of the acetylcholine receptor by selective T1 NMR relaxation.

Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence alpha 184-200 from the Torpedo californica receptor. This construct binds alpha-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control [Aronheim, A., Eshel, Y., Mosckovitz, R., & Gershoni, J. M. (1988) J. Biol. Chem. 263, 9933-9937]. In this study agonist binding to alpha 184-200 is demonstrated by monitoring the T1 relaxation of the ligand's protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with alpha-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified alpha 184-200 constructs, respectively. The KD values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while KD's with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain alpha 184-200 may comprise a major component of the cholinergic agonist site.

Acetylcholine↗
PubMed1988

Characterization of the binding of alpha-bungarotoxin to bacterially expressed cholinergic binding sites.

Bacterially expressed cDNA fragments of the alpha-subunit of the nicotinic acetylcholine receptor previously have been shown to bind alpha-bungarotoxin (Gershoni, J. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4318-4321). Here, a novel system has been developed in which totally synthetic alpha-bungarotoxin binding sites are expressed in Escherichia coli transformants. The amino acid sequences, alpha 184-200 and alpha 184-196 of the Torpedo californica alpha-subunit of the nicotinic acetylcholine receptor were expressed as trpE fusion proteins via the expression vector pATH2 and a method for the enrichment of these fusion proteins is described. Quantitative analysis of toxin binding to the recombinant binding sites demonstrates that they bind toxin with affinities of KD = 2.5 X 10(-7) and 4.7 X 10(-6) M, respectively. Furthermore, the pharmacological profile of alpha 184-200 qualitatively reflects that of the intact receptor. These data not only indicate that the area of alpha 184-200 is an essential element of the cholinergic binding site but that residues alpha 197-200 contribute a point of contact between the receptor and alpha-bungarotoxin.

Amino Acid Sequence↗
PubMed1988

Three possible disulfides in the acetylcholine receptor alpha-subunit.

The cysteinyl residues of the acetylcholine receptor alpha-subunit of Torpedo californica were analyzed. All seven cysteines could be accounted for. Three possible disulfide bridges and one unpaired cysteine were indicated. The disulfide linkages were as follows: Cys128 to Cys142; Cys192 to Cys193; Cys412 to Cys418 (Cys222 is unpaired). The identification of cysteinyl residues was accomplished by a modified protein blot procedure. Cysteinyl residues of intact nicotinic acetylcholine receptor were selectively biotinylated with 3-(N-maleimidopropionyl)biocytin and subsequently detected by the 125I-labeled avidin overlay of blotted Staphylococcus aureus V8 proteolyzed alpha-subunits. Two pairs of cysteines (Cys128/Cys142 and Cys412/Cys418) could be demonstrated only after Na(BH4) reduction of the acetylcholine receptor. Cysteine residues 192 and 193 are particularly sensitive to reduction; 0.1 mM dithiothreitol is sufficient.

Animals↗
PubMed1988

Molecular decoys: ligand-binding recombinant proteins protect mice from curarimimetic neurotoxins.

Mimic ligand-binding sites of the nicotinic acetylcholine receptor bind d-tubocurarine and alpha-bungarotoxin in vitro. Injection of such binding sites into mice could act as molecular decoys in vivo, providing protection against toxic ligands. This hypothesis of molecular "decoyance" has been tested in greater than 250 mice. Bacterially produced cholinergic binding sites provided a 2-fold increase in the survival rate of animals challenged with curarimimetic neurotoxins. Possible considerations for decoy designs and their applications are discussed.

Animals↗
PubMed1987

Biotin binding to avidin. Oligosaccharide side chain not required for ligand association.

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.

Avidin↗
PubMed1987

Localization of azidophencyclidine-binding site on the nicotinic acetylcholine receptor alpha-subunit.

Nicotinic acetylcholine receptors in receptor-rich membranes from Torpedo californica and from T. marmorata electric tissue were photolabeled with the non-competitive inhibitor [3H]azidophencyclidine. The receptor subunits were separated on SDS-polyacrylamide gels and the alpha-subunits recovered from the gel, were subjected to Staphylococcus aureus V8 protease cleavage. The proteolytic fragments were resolved by SDS-polyacrylamide gel electrophoresis and were identified on protein blots by 125I-labeled alpha-bungarotoxin binding and by staining with concanavalin A. The site of specific azidophencyclidine labeling has been localized to the V8-18 kDa fragment which binds toxin. Labeling of the V8-18 kDa fragment was observed in the absence and in the presence of carbamylcholine. This was found for both the species of Torpedo used here.

Affinity Labels↗
PubMed1987

Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants.

Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots.

Animals↗
PubMed1986

Identification of cell surface glycoproteins by periodate-alkaline phosphatase hydrazide.

A novel method for the detection of cell surface glycoconjugates has been developed. Cells are subjected to mild surface oxidation of vicinal hydroxyls with sodium periodate. Afterward, cellular proteins are resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and then probed with alkaline phosphatase hydrazide. The technique is sensitive, reproducible, and inexpensive. It obviates the need for radiolabeled NaBH4 and the subsequent processing of polyacrylamide gels for fluorography. Results are easily obtained in a matter of a few hours.

Alkaline Phosphatase↗
PubMed1986

Alpha-bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots.

The binding of [125I]iodo-alpha-bungarotoxin [( 125]alpha-BuTX) to the dissociated alpha-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified 'protein-blot' analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [125I]alpha-BuTX. We report here the use of the protein-blotting technique to detect the alpha-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (Mr greater than or equal to 200,000 and greater than or equal to 120,000) alpha-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [125I]alpha-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the alpha-subunit (Mr = 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrate the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.

Animals↗
PubMed1986

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor.

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.

Animals↗
PubMed1986

Identification of glycoproteins that are receptors for peanut agglutinin on immature (cortical) mouse thymocytes.

Binding of peanut agglutinin is being widely used as a marker for immature mouse thymocytes and for the separation of these cells from the mature thymocytes. Two cell surface glycoproteins that bind peanut agglutinin were detected on unfractionated as well as immature thymocytes by lectin overlay and affinity chromatography: one of Mr between 170 000 and 180 000, and the other, a minor component, of Mr 110000, both of which are partially sialylated. No receptors for peanut agglutinin were detected on the mature cells, whereas desialylation experiments revealed the presence of a glycoprotein of Mr 110000. These findings were corroborated by electrophoretic analysis of cell surface glycoproteins of the isolated thymocyte subpopulations labeled in their carbohydrate moieties.

Animals↗