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Baruch I Kanner - One of the best experts on this subject based on the ideXlab platform.
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the interaction of the γ aminobutyric acid transporter gat 1 with the neurotransmitter is selectively impaired by sulfhydryl modification of a conformationally sensitive Cysteine Residue engineered into extracellular loop iv
Journal of Biological Chemistry, 2003Co-Authors: Elia Zomot, Baruch I KannerAbstract:The (Na+ + Cl-)-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by Cysteine scanning mutagenesis. Fourteen Residues of this loop have been replaced by Cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible Cysteine Residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 degrees C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.
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the interaction of the γ aminobutyric acid transporter gat 1 with the neurotransmitter is selectively impaired by sulfhydryl modification of a conformationally sensitive Cysteine Residue engineered into extracellular loop iv
Journal of Biological Chemistry, 2003Co-Authors: Elia Zomot, Baruch I KannerAbstract:The (Na+ + Cl–)-coupled γ-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by Cysteine scanning mutagenesis. Fourteen Residues of this loop have been replaced by Cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible Cysteine Residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 °C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.
Toru Hisabori - One of the best experts on this subject based on the ideXlab platform.
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oxidation of a Cysteine Residue in elongation factor ef tu reversibly inhibits translation in the cyanobacterium synechocystis sp pcc 6803
Journal of Biological Chemistry, 2016Co-Authors: Rayakorn Yutthanasirikul, Takuya Ueda, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru HisaboriAbstract:Abstract Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G (EF-G) has been identified as a target of oxidation by ROS. In the present study, we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu, as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys82, a single Cysteine Residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of more than 30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling EF-G, EF-Tu appears to be sensitive to ROS via oxidation of a Cysteine Residue and its inactivation might be reversed in a redox-dependent manner.
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Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803
The Journal of biological chemistry, 2016Co-Authors: Rayakorn Yutthanasirikul, Takuya Ueda, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru HisaboriAbstract:Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single Cysteine Residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a Cysteine Residue, and its inactivation might be reversed in a redox-dependent manner.
Elia Zomot - One of the best experts on this subject based on the ideXlab platform.
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the interaction of the γ aminobutyric acid transporter gat 1 with the neurotransmitter is selectively impaired by sulfhydryl modification of a conformationally sensitive Cysteine Residue engineered into extracellular loop iv
Journal of Biological Chemistry, 2003Co-Authors: Elia Zomot, Baruch I KannerAbstract:The (Na+ + Cl-)-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by Cysteine scanning mutagenesis. Fourteen Residues of this loop have been replaced by Cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible Cysteine Residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 degrees C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.
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the interaction of the γ aminobutyric acid transporter gat 1 with the neurotransmitter is selectively impaired by sulfhydryl modification of a conformationally sensitive Cysteine Residue engineered into extracellular loop iv
Journal of Biological Chemistry, 2003Co-Authors: Elia Zomot, Baruch I KannerAbstract:The (Na+ + Cl–)-coupled γ-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by Cysteine scanning mutagenesis. Fourteen Residues of this loop have been replaced by Cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible Cysteine Residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 °C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.
Rayakorn Yutthanasirikul - One of the best experts on this subject based on the ideXlab platform.
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oxidation of a Cysteine Residue in elongation factor ef tu reversibly inhibits translation in the cyanobacterium synechocystis sp pcc 6803
Journal of Biological Chemistry, 2016Co-Authors: Rayakorn Yutthanasirikul, Takuya Ueda, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru HisaboriAbstract:Abstract Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G (EF-G) has been identified as a target of oxidation by ROS. In the present study, we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu, as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys82, a single Cysteine Residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of more than 30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling EF-G, EF-Tu appears to be sensitive to ROS via oxidation of a Cysteine Residue and its inactivation might be reversed in a redox-dependent manner.
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Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803
The Journal of biological chemistry, 2016Co-Authors: Rayakorn Yutthanasirikul, Takuya Ueda, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru HisaboriAbstract:Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single Cysteine Residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a Cysteine Residue, and its inactivation might be reversed in a redox-dependent manner.
Yukako Hihara - One of the best experts on this subject based on the ideXlab platform.
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oxidation of a Cysteine Residue in elongation factor ef tu reversibly inhibits translation in the cyanobacterium synechocystis sp pcc 6803
Journal of Biological Chemistry, 2016Co-Authors: Rayakorn Yutthanasirikul, Takuya Ueda, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru HisaboriAbstract:Abstract Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G (EF-G) has been identified as a target of oxidation by ROS. In the present study, we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu, as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys82, a single Cysteine Residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of more than 30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling EF-G, EF-Tu appears to be sensitive to ROS via oxidation of a Cysteine Residue and its inactivation might be reversed in a redox-dependent manner.
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Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803
The Journal of biological chemistry, 2016Co-Authors: Rayakorn Yutthanasirikul, Takuya Ueda, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru HisaboriAbstract:Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single Cysteine Residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a Cysteine Residue, and its inactivation might be reversed in a redox-dependent manner.
