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Journal of Receptor Research 1989Homogenates from dog cerebellum were fractionated using sucrose gradient centrifugation. The [3H]inositol 1,4,5-trisphosphate binding and the glucose 6-phosphatase...
Homogenates from dog cerebellum were fractionated using sucrose gradient centrifugation. The [3H]inositol 1,4,5-trisphosphate binding and the glucose 6-phosphatase activities were found to co-purify. The binding was saturable and had high affinity (Bmax = 44 pmol/mg protein, Kd = 116 nM). Selective chemical modification was used to examine amino acid residues of the microsomal receptor that might be critical for the binding of inositol trisphophate. Sulfhydryl reagents, p-chloromercuricphenyl sulfonic acid. eosin 5-maleimide, N-ethyl maleimide and fluorescein 5-maleimide were found to be highly potent inhibitors of the binding with half-maximal inhibition occurring at about 20 microM, 70 microM, 1 mM, and 0.1 mM, respectively. The inhibition was specific since the presence of 10 microM of inositol trisphosphate during the reaction completely protected against the inhibition by these reagents. These results suggest that sulfhydryl group is essential for inositol trisphosphate binding to its receptor.
Topics: Animals; Binding Sites; Calcium Channels; Cerebellum; Dogs; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Inositol Phosphates; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Sugar Phosphates; Sulfhydryl Reagents
PubMed: 2545875
DOI: 10.3109/10799898909066051 -
FEBS Letters Jul 1980
Topics: Ammonia-Lyases; Dithioerythritol; Hydroxymethylbilane Synthase; Kinetics; Plants; Structure-Activity Relationship; Sulfhydryl Reagents
PubMed: 7409146
DOI: 10.1016/0014-5793(80)80643-0 -
Analytical Biochemistry May 1982
New reagents for the introduction of the thiomethyl group at sulfhydryl residues of proteins with concomitant spectrophotometric titration of the sulfhydryls: methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide.
Topics: Chemical Phenomena; Chemistry; Methylation; Peptides; Proteins; Pyridines; Spectrophotometry, Ultraviolet; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 7114445
DOI: 10.1016/0003-2697(82)90281-0 -
Molecular Pharmacology Jul 1978
Topics: Animals; Brain; Ethylmaleimide; Kinetics; Membranes; Mercuribenzoates; Metals; Rats; Receptors, Cholinergic; Receptors, Muscarinic; Sulfhydryl Reagents
PubMed: 683175
DOI: No ID Found -
The Journal of Biological Chemistry May 1948
Topics: Sulfhydryl Reagents
PubMed: 18914057
DOI: No ID Found -
Proceedings of the National Academy of... Jun 1994The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two alpha-helices, lying within the membrane, connected...
The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two alpha-helices, lying within the membrane, connected by a short interhelical loop of four amino acids (residues 349-352). To investigate the validity and implications of this model, we singly mutated each of these amino acids to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and added to the trans compartment sulfhydryl-specific reagents [methanethiosulfonate derivatives (MTS-ER)] that introduce a positive or negative charge to reacted cysteines. The introduction of a positive charge at residue 351 or 352 (through the MTS-ER reactions) resulted in a step decrease in single-channel conductance, whereas the introduction of a negative charge resulted in a step increase. The opposite sign of these effects indicates the predominantly electrostatic nature of the phenomenon and implies that residues 351 and 352 lie close to the channel entrance. The same reactions at residue 350 resulted in very little change in channel conductance but instead changed the character of the natural rapid flickering of the channel between open and closed states to one in which the channel spent more time in the closed state; this may have resulted from the group introduced at position 350 acting as a tethered channel blocker. The MTS derivatives had no effect on channels containing a cysteine at position 349, suggesting that this residue faces away from the channel entrance. We propose that the step changes in conductance or flickering pattern result from the chemical reaction of one MTS-ER molecule with one cysteine, and thus a bimolecular chemical reaction is being witnessed at the single molecule level. From the distribution of waiting times between the appearance (i.e., the opening) of a channel and the step change in its conductance or flickering pattern, we can calculate a pseudo-first-order rate constant, which can then be converted to a second-order rate constant, for the chemical reaction.
Topics: Amino Acid Sequence; Diphtheria Toxin; Electric Conductivity; In Vitro Techniques; Ion Channels; Lipid Bilayers; Molecular Sequence Data; Mutagenesis, Site-Directed; Structure-Activity Relationship; Sulfhydryl Reagents
PubMed: 7515494
DOI: 10.1073/pnas.91.12.5272 -
The American Journal of Physiology Jul 1986The lateral giant axons of the crayfish nerve cord are composed of segments contributed by each ganglion, which are electrotonically coupled by way of gap junctions. We...
The lateral giant axons of the crayfish nerve cord are composed of segments contributed by each ganglion, which are electrotonically coupled by way of gap junctions. We have investigated the involvement of protein residues in regulating the resistance of crayfish junctional channels by determining effects of group-specific protein reagents. When applied to well-coupled axons, the sulfhydryl group reagents N-ethylmaleimide (NEM) and diamide uncoupled the segments; junctional resistance (Rj) was increased without changing membrane resistance or axoplasmic pH (pHi). The uncoupling produced by NEM could be reversed by alkalinization of the cytoplasm (addition of ammonium chloride to the external medium). Another sulfhydryl reagent (p-chloromercuribenzoic acid) increased Rj to a lesser extent. A disulfide reagent and three amino and three carboxyl group reagents had no effect on the Rj of these axons. The effect of group-specific reagents on partially uncoupled axons was tested by applying the drugs to axons previously exposed to weak acids. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline recoupled partially uncoupled axons by decreasing Rj and prevented subsequent uncoupling of the junction by low pHi. Another carboxyl group reagent, as well as sulfhydryl and amino group reagents, either had no effect or uncoupled the axons further by increasing Rj. These experimental results suggest that amino acid residues, possibly containing carboxyl and sulfhydryl groups, control the opening and closing of junctional channels and may thus be associated with the channels' active sites.
Topics: Amino Acids; Animals; Astacoidea; Axons; Connexins; Electric Conductivity; Indicators and Reagents; Intercellular Junctions; Membrane Proteins; Sulfhydryl Reagents
PubMed: 3014886
DOI: 10.1152/ajpcell.1986.251.1.C99 -
Chemical & Pharmaceutical Bulletin Feb 1964
Topics: Benzimidazoles; Chemistry, Pharmaceutical; Imides; Indicators and Reagents; Maleimides; Research; Spectrum Analysis; Sulfhydryl Compounds; Sulfhydryl Reagents
PubMed: 14126740
DOI: 10.1248/cpb.12.127 -
Methods in Cell Biology 2010Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule... (Review)
Review
Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule polymerization. 5',5'-Dithiobis(2-nitrobenzoate) (DTNB) reacts with free and accessible sulfhydryls and stoichiometrically produces a detectable product, which allows an exact measurement of reacted thiols. Since binding of small ligands may alter conformational dynamics, it may also affect the reactivity of thiols on tubulin. Differences in DTNB reactivity with thiols upon ligand binding can therefore be used to deduce binding characteristics. We will describe two methods that use tubulin cysteine reactivity with DTNB in the presence of drug to define ligand-binding characteristics.
Topics: Animals; Drug Evaluation, Preclinical; Humans; Ligands; Protein Binding; Sulfhydryl Compounds; Sulfhydryl Reagents; Tubulin; Tubulin Modulators
PubMed: 20466146
DOI: 10.1016/S0091-679X(10)95021-8 -
Bioorganic & Medicinal Chemistry Letters Feb 2003The preparation of several model vinylsulfamides is described. Their excellent selective reactivity towards sulfhydryl group with regards to amino group has been...
The preparation of several model vinylsulfamides is described. Their excellent selective reactivity towards sulfhydryl group with regards to amino group has been demonstrated by the kinetics study between a model vinylsulfamide and cysteine or lysine at different pHs.
Topics: Alkylating Agents; Ethylenes; Half-Life; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Proteins; Sulfhydryl Compounds; Sulfhydryl Reagents; Sulfonamides
PubMed: 12565934
DOI: 10.1016/s0960-894x(02)00950-2