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Acta Neurobiologiae Experimentalis 1997In the present study the functional role of SH groups in the Ca(2+)-independent K(+)-selective channel activity in the membrane of bovine adrenal gland chromaffin...
In the present study the functional role of SH groups in the Ca(2+)-independent K(+)-selective channel activity in the membrane of bovine adrenal gland chromaffin granules has been studied. Ionic channel activity has been estimated using 86Rb+, a K+ analogue, flux measurements. The inhibition of chromaffin granules K+ channel by SH modifying agents, such as N-ethylmaleimide, mersalyl and phenylarsenoxide, is described.
Topics: Adrenal Medulla; Animals; Biological Transport; Cattle; Chromaffin Cells; Potassium; Potassium Channels; Sulfhydryl Reagents
PubMed: 9519550
DOI: 10.55782/ane-1997-1242 -
Trends in Pharmacological Sciences Nov 1995NMDA receptors play a central role in neuronal plasticity and in several pathological situations. Transient activation of this receptor triggers long-term potentiation,... (Review)
Review
NMDA receptors play a central role in neuronal plasticity and in several pathological situations. Transient activation of this receptor triggers long-term potentiation, whereas sustained activation leads to cell death. Evidence for control of this activity by a redox site in cell cultures, brain tissues and in recombinant NMDA receptors are discussed by Henri Gozlan and Yehezkel Ben-Ari. The characteristics of this modulation and the consequences of redox state modifications on NMDA-mediated events are examined in vitro under physiological and pathological conditions. Since metabolic disorders enhance NMDA receptor function, the redox site could constitute a new target for selectively preventing in vivo the deleterious consequences of overactivation without blocking neuronal plasticity mediated by NMDA receptors.
Topics: Alkylation; Animals; Ascorbic Acid; Brain; Cells, Cultured; Epilepsy; Free Radicals; In Vitro Techniques; Ischemia; Neuronal Plasticity; Oxidation-Reduction; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Sulfhydryl Compounds; Sulfhydryl Reagents; Synaptic Transmission; Xenopus laevis
PubMed: 8578605
DOI: 10.1016/s0165-6147(00)89077-x -
Experientia Feb 1975
Topics: Animals; Binding Sites; Calcium; Cell Membrane; Dose-Response Relationship, Drug; Magnesium; Mercury; Microsomes, Liver; Rats; Sulfhydryl Reagents
PubMed: 1112355
DOI: 10.1007/BF01990705 -
Analytical Biochemistry May 1978
Topics: Energy Transfer; Fluorescence; Sulfhydryl Reagents
PubMed: 655390
DOI: 10.1016/0003-2697(78)90346-9 -
Journal of Visualized Experiments : JoVE Mar 2019Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet...
Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent - dubbed 'PODS' - as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.
Topics: Animals; Humans; Immunoconjugates; Maleimides; Mice; Sulfhydryl Reagents; Trastuzumab
PubMed: 30907883
DOI: 10.3791/59063 -
Biomembranes 1975The role of reactive SH groups (presumably in proteins) of the apical plasma membrane in transepithelial Na+ transport was studied in the isolated urinary bladder of the...
The role of reactive SH groups (presumably in proteins) of the apical plasma membrane in transepithelial Na+ transport was studied in the isolated urinary bladder of the toad. On the basis of assays for TCA-soluble SH compounds (e.g., glutathione, methionine), PCMB, PCMPS, NTCB, and DTNB did not penetrate the intracellular compartment from the luminal media either in control or vasopressin-treated bladders. In contrast, PCMB from the serosal side and NEM from the luminal side titrated significant fractions of the TCA-soluble SH compounds. We conclude, therefore, the PCMB, PCMPS, NTCB, and DTNB are suitable reagents for studies on the physiological properties of apical plasma membrane SH groups. Titration of apical membrane SH groups with PCMPS, NTCB, and DTNB revealed heterogeneity in functional responses: PCMPS and NTCB elicited transient, 25-60% increases in SCC. In substrate-free media, pretreatment with these reagents inhibited the increase in SCC produced by vasopressin or cyclic AMP (+ theophylline). In glucose-enriched media, the responses to combinations of vasopressin and PCMPS or NTCB were additive, implying activation via parallel pathways. Simultaneous addition of vasopressin or cyclic AMP (+ theophylline) and NTCB resulted in marked synergism, presumably as a result of unmasking of SH groups by the the hormone (or the intermediate). These results suggest that basal Na+ transport is regulated in part by SH compounds in the apical membrane that are distinct, although not necessarily different in kind, from those involved in the response to vasopressin.
Topics: Amiloride; Animals; Biological Transport, Active; Bufo marinus; Cell Membrane; Cyclic AMP; Epithelium; Female; Sodium; Sulfhydryl Reagents; Theophylline; Urinary Bladder; Vasopressins
PubMed: 164961
DOI: 10.1007/978-1-4684-7668-2_4 -
Journal of Biochemistry Jul 1981L-Galactonolactone oxidase was found to be inactivated by various sulfhydryl reagents: the order of inactivation rate was HgCl2 greater than p-chloromercuribenzoate...
L-Galactonolactone oxidase was found to be inactivated by various sulfhydryl reagents: the order of inactivation rate was HgCl2 greater than p-chloromercuribenzoate greater than 4,4'-dipyridyldisulfide greater than 2,2'-dipyridyldisulfide greater than 5,5'-dithio-bis-(2-nitrobenzoate) greater than N-ethylmaleimide greater than iodoacetamide. The inactivation by 4,4'dipyridyldisulfide was studied in detail, and it was found that the maximum degree of inactivation attained with increasing reagent concentration was 93%, and that the kinetics of inactivation was first order with respect to the reagent concentration. The pH dependence of the second-order rate constant of the inactivation revealed that sulfhydryl group with a pKa of approximately 9.8 was involved in the inactivation process. The value of pKa is high compared with that of low-molecular weight thiols, indicating that the ionization of the sulfhydryl group is affected by the electric field of a negatively charged group located in its vicinity. The values of Km and Vmax for the L-galactonolactone oxidase reaction did not change greatly around pH 9.8. This finding is consistent with the view that the sulfhydryl group does not participate in the catalytic process. The velocity of inactivation in the presence of substrate was considerably greater than that observed in its absence. It appears that the sulfhydryl group becomes more reactive during the catalytic cycle of the enzyme. It was also noted that the absorption spectrum of the flavin prosthetic group (the oxidized form) was modified by adding p-chloromercuribenzoate.
Topics: Chloromercuribenzoates; Hydrogen-Ion Concentration; Kinetics; Mercaptoethanol; Plants; Protein Binding; Spectrophotometry; Structure-Activity Relationship; Sugar Alcohol Dehydrogenases; Sulfhydryl Reagents; p-Chloromercuribenzoic Acid
PubMed: 7287686
DOI: 10.1093/oxfordjournals.jbchem.a133467 -
Methods in Molecular Biology (Clifton,... 2019Abnormal protein-protein interactions (PPIs) are the basis of multiple diseases, and the large and shallow PPI interfaces make the target "undruggable" for traditional...
Abnormal protein-protein interactions (PPIs) are the basis of multiple diseases, and the large and shallow PPI interfaces make the target "undruggable" for traditional small molecules. Peptides, emerging as a new therapeutic modality, can efficiently mimic PPIs with their large scaffolds. Natural peptides are flexible and usually have poor serum stability and cell permeability, features that limit their further biological applications. To satisfy the clinical application of peptide inhibitors, many strategies have been developed to constrain peptides in their bioactive conformation. In this report, we describe several classic methods used to constrain peptides into a fixed secondary structure which could significantly improve their biophysical properties.
Topics: Amides; Biophysical Phenomena; Circular Dichroism; Crystallography, X-Ray; Hydrocarbons; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Peptides; Protein Conformation, alpha-Helical; Protein Stability; Protein Structure, Secondary; Solid-Phase Synthesis Techniques; Sulfhydryl Reagents
PubMed: 31134570
DOI: 10.1007/978-1-4939-9504-2_7 -
The Journal of Biological Chemistry Apr 1989Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a...
Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activity. A peculiar feature of these protein thiol groups is that they react nonidentically with a number of thiol blocking reagents, i.e. iodacetamide, bromopyruvate, N-ethylmaleimide, and 1-chloro-2,4-dinitrobenzene. Also many disulfides react with one thiol group 5- to 10-fold more rapidly than with the other. The two mixed disulfides so formed also have different rates of reactivation by dithiothreitol. All the structural and kinetic data reported in this paper indicate a nonsymmetrical association of two identical subunits, or alternatively heterodimeric structure with subunits of very similar charge and size.
Topics: Amino Acids; Animals; Disulfides; Erythrocytes; Glutathione Transferase; Horses; Kinetics; Macromolecular Substances; Substrate Specificity; Sulfhydryl Reagents
PubMed: 2925613
DOI: No ID Found -
Journal of Biochemical and Biophysical... Mar 1992In this study we have searched for sulfhydryl reagents which can be radiolabeled and detect minute quantities of SH-proteins. Iodoacetamidotyramine reacts with...
In this study we have searched for sulfhydryl reagents which can be radiolabeled and detect minute quantities of SH-proteins. Iodoacetamidotyramine reacts with sulfhydryls at a low rate, having a pseudo-first order rate constant, kappa obs = 3 +/- 0.2 M-1 s-1, at neutral pH. In contrast, N-ethylmaleimide-containing reagents, such as tyrosine-MIB and tyramine-MIB were three orders of magnitude more reactive in alkylating sulfhydryls. Pseudo-first order rate constants, kappa obs, were in the range of 5200-5700 M-1 s-1. Therefore, a simple and convenient procedure was designed for the synthesis and the radioactive labeling of tyramine-MIB. Simplification was attained by virtue of the specific-'affinity' adsorption of [125I]tyramine-MIB (and not the other intermediates) to small Sephadex G-10 column and its elution with ethanol. [125I]Tyramine-MIB was stable for weeks in dried form and for hours in acidic to neutral aqueous solutions. The reagent, when radiolabeled to high specific activity (0.5 Ci/mumol), detected sulfhydryl proteins at concentrations as low as 1-10 pM. The applicability of the reagent in studying biological systems was demonstrated by adding it to intact adipocytes and the consequent labeling of a single protein with an apparent Mr = 32,000, which is most likely an externally oriented surface plasma membrane SH-protein. [125I]Tyramine-MIB reactivity and sensitivity exceeds that of protein-tyrosyl radioiodination by the chloramine-T procedure and is expected to assist in studying minute quantities of SH-proteins.
Topics: Adipose Tissue; Animals; Indicators and Reagents; Iodine Radioisotopes; Membrane Proteins; Rats; Structure-Activity Relationship; Succinimides; Sulfhydryl Reagents; Tyramine
PubMed: 1560185
DOI: 10.1016/0165-022x(92)90050-k