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The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor 总被引:1,自引:0,他引:1
W Tienrungroj S Meshinchi E R Sanchez S E Pratt J F Grippo A Holmgren W B Pratt 《The Journal of biological chemistry》1987,262(15):6992-7000
Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor complex that accompanies transformation to the DNA-binding state. When steroid-receptor complexes that are prebound to DNA-cellulose are exposed to MMTS, the steroid rapidly dissociates, but the receptor remains bound to DNA. Thus, the presence of steroid is not required for the receptor to remain bound to DNA in a high affinity manner. Treatment of cytosol containing transformed glucocorticoid-receptor complexes at 0 degrees C with 20 mM hydrogen peroxide also inactivates the DNA-binding activity of the receptor. The peroxide-induced inactivation is reversed by DTT. Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25 degrees C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. The presence of peroxide during heating of the cytosol also prevents dissociation of the receptor complex as assayed both by reduction in sedimentation value of the receptor and by dissociation of the 90-kDa heat shock protein from the steroid-binding protein. These results strongly suggest that critical sulfur moieties in the receptor complex must be in a reduced form for the temperature-mediated dissociation of the receptor to occur. 相似文献
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A role of sulfhydryl groups in imidazoleacetate monooxygenase 总被引:2,自引:0,他引:2
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Benextramine, a tetramine disulfide, irreversibly inhibits neuropeptide Y (NPY) binding to the 50-kDa Y2 NPY receptor in bovine hippocampus (Li, W., MacDonald, R. G., and Hexum, T. D. (1991) Eur. J. Pharmacol. 207, 89-91). Evidence is presented that this inhibition occurs through a thiol-disulfide exchange. Treatment of bovine hippocampal membranes with benextramine inhibited NPY affinity cross-linking to the 50-kDa receptor. This inhibition of labeling was not affected by washing the membranes, but could be completely reversed by the addition of several thiol reducing reagents, including reduced glutathione, beta-mercaptoethanol, and cysteine. Benextramine inhibited 70% of NPY-specific labeling and was much more effective than other sulfhydryl reactive agents, such as oxidized glutathione, cystamine, and 5,5'-dithio-bis(2-nitrobenzoic acid). Furthermore, the sulfhydryl-modifying agents N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid specifically decreased NPY affinity labeling. Finally, NPY labeling of the 50-kDa receptor was reduced by the heavy metal ions Zn2+, Cu2+, and Hg2+. Preincubation with NPY prevented Y2 receptors from being inactivated by either 400 microM N-ethylmaleimide or 1 mM benextramine. These results suggest that one or more benextramine-sensitive sulfhydryl groups on the Y2 receptor are important for NPY binding activity. 相似文献
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The role of sulfhydryl groups in the activity of type A botulinum toxin 总被引:10,自引:0,他引:10
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The binding of GABA to postsynaptic receptors was studied in the cerebral and cerebellar cortex, caudate nucleus, putamen, pallidum and substantia nigra from autopsy brains of 12 parkinsonian patients and 11 controls. GABA receptor binding in the substantia nigra was significantly decreased in the parkinsonian brain. In the other brain regions, however, GABA binding was unchanged. There was no correlation between GABA binding and sex, age, duration or severity of the disease. The results suggest the involvement of nigral GABA receptor in Parkinson's disease. 相似文献
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K. Vasudeva Rao 《Development genes and evolution》1969,163(2):161-165
Summary The teratogenic effects on chick embryos of chloroacetophenone (CAP), a specific sulfhydryl blocking agent, are known to be reversible by a subsequent treatment with cysteine. However, cysteamine, the decarboxylation product of cysteine has been found to be unable to ameliorate the syndrome caused by CAP. It is suggested that CAP may act by binding the sulfhydryl of free cysteine in the cells of the organizer, thus indicating that cysteine may act as an inducing stimulus in primary organizer action. Post-nodal pieces of chick blastoderm exposed to the action of cysteine for 6 hours show differentiation of axial embryonic structures. Cysteamine has no such stimulatory effects on post-nodal pieces. This finding seems to further support the conclusion that cysteine may act as an inducing stimulus in primary organizer action.
The author is grateful to Prof. B. R.Seshachar for constant encouragement and facilities for work. This work was supported by financial assistance from the Council of Scientific and Industrial Research, Government of India. 相似文献
Zusammenfassung Die teratogenen Effekte von Chloroacetophenon auf Hühnchenembryonen sind bekannterweise reversibel durch Nachbehandlung mit Cystein. CAP ist ein Agens, das Sulphhydrylgruppen blockiert. Cysteamine revertiert die teratogenen Effekte nicht. Es wird vorgeschlagen, da\ CAP die Sulphhydrylgruppen freien Cysteins in den Zellen des Organisators blockiert, was hei\en würde, da\ Cystein ein induzierender Stimulus in der Organisatorwirkung wäre. Wenn post-nodale Stücke von Hühnchenblastoderm während 6 Std der Wirkung von Cystein ausgesetzt werden, entwickeln diese Axialelemente. Cysteamin zeigt diesen Effekt nicht. Diese Resultate stützen die Annahme, da\ Cystein als Induktor im primären Organisator wirken könnte.
The author is grateful to Prof. B. R.Seshachar for constant encouragement and facilities for work. This work was supported by financial assistance from the Council of Scientific and Industrial Research, Government of India. 相似文献
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Sulfhydryl groups of Escherichia coli DNA-dependent RNA polymerase were chemically modified with alkylating and mercuric-containing compounds. Iodoacetic acid and iodoacetamide were shown not to affect the enzymatic activity, whereas N-ethylmaleimide and mercuric-containing compounds completely inhibit the RNA synthesis. RNA polymerase modified with mercuric ions looses the ability of binding with promoter--containing DNA fragments. Moreover, mercuric ions inhibit the RNA elongation stage. Suggestion is made the Cys residues of RNA polymerase play a key role in double-stranded DNA unwinding. It is shown that SH-groups of beta- and beta'-subunits participate in the binding with double-stranded fragments of DNA. 相似文献
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An endogenous inhibitor of GABA receptor binding was prepared from synaptic membrane of rat brain with 0.05% Triton X-100. The endogenous inhibitor was competitive with GABA for GABA binding sites. The inhibition of GABA receptor binding by the endogenous inhibitor was blocked by the allosteric effect of diazepam. In the presence of diazepam, specific [3H]GABA binding was greater in a medium containing the endogenous inhibitor than in one containing an equal inhibitory potency of GABA, whereas there was no difference in the absence of diazepam. This indicated that the endogenous inhibitor was not GABA itself. 相似文献
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The mitochondrial inner membrane lost its selectivity for the transport of solutes after reaction of hydrophobic sulfhydryl groups with alkylating agents (maleimide derivatives). The nature of the thiol reagent-induced membrane perturbations was investigated. Modifications of the interactions between membrane components after treatment with thiol reagents were assessed by measuring the binding parameters of 1-anilinonaphtalene-8-sulfonate. An enhancement (about 50%) of the fluorescence intensity, a weak increase of the number of binding sites, and a decrease of the apparent dissociation constant were observed. However, no significant modification of the net surface charge was detected. The osmotic behavior of mitochondria in hypotonic solutions of sucrose was altered after thiol modification. The outer membrane did not seem to influence the matricial volume expansion when thiols were alkylated. After swelling in an isotonic solution of permeant ions, N-butylmaleimide-treated mitochondrial lost one-half of their malate dehydrogenase content, whereas fumarase and glutamate dehydrogenase did not leave the matrix space. Addition of polyethylene glycol of molecular weight below 6000 to swollen mitochondria induced a rapid but transient shrinkage. In swollen mitochondria, the above results indicate a possible holes formation in the membrane structure. The size of these holes was estimated to be about 3 nm. This process which required the presence of the outer membrane, was favored by increasing the temperature and was antagonized by specific effectors of the adenine nucleotide translocator. 相似文献
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A gain-of-function mutation in the GABA receptor produces synaptic and behavioral abnormalities in the mouse 总被引:3,自引:0,他引:3
Homanics GE Elsen FP Ying SW Jenkins A Ferguson C Sloat B Yuditskaya S Goldstein PA Kralic JE Morrow AL Harrison NL 《Genes, Brain & Behavior》2005,4(1):10-19
In mammalian species, inhibition in the brain is mediated predominantly by the activation of GABAA receptors. We report here changes in inhibitory synaptic function and behavior in a mouse line harboring a gain-of-function mutation at Serine 270 (S270) in the GABAA receptor alpha1 subunit. In recombinant alpha1beta2gamma2 receptors, replacement of S270 by Histidine (H) results in an increase in sensitivity to gamma-aminobutyric acid (GABA), and slowing of deactivation following transient activation by saturating concentrations of GABA. Heterozygous mice expressing the S270H mutation are hyper-responsive to human contact, exhibit intention tremor, smaller body size and reduced viability. These mice also displayed reduced motor coordination, were hypoactive in the home cage, but paradoxically were hyperactive in a novel open field environment. Heterozygous knockin mice of both sexes were fertile but females failed to care for offspring. This deficit in maternal behavior prevented production of homozygous animals. Recordings from brain slices prepared from these animals revealed a substantial prolongation of miniature inhibitory postsynaptic currents (IPSCs) and a loss of sensitivity to the anesthetic isoflurane, in neurons that express a substantial amount of the alpha1 subunit. The results suggest that the biophysical properties of GABAA receptors are important in determining the time-course of inhibition in vivo, and suggest that the duration of synaptic inhibition is a critical determinant that influences a variety of behaviors in the mouse. 相似文献
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The condensation of retinene1 with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene1 with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene1. Its action in this system is confined to the —SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the —SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene1 molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene1. In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general. 相似文献