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Italo Stipani - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5′-Phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by Pyridoxal 5′-Phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5′-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC_50 of 0.5 m M , competed with the substrate for binding to the oxoglutaratecarrier ( K _ i = 0.4 m M ). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal5′-Phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [^3H]Pyridoxal 5′-Phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by Pyridoxal 5′-Phosphate. These results indicate that Pyridoxal 5′-Phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.
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Inactivation of the reconstituted oxoglutarate carrier from bovine heart mitochondria by Pyridoxal 5'-Phosphate.
Journal of bioenergetics and biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5'-Phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by Pyridoxal 5'-Phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal 5'-Phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]Pyridoxal 5'-Phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by Pyridoxal 5'-Phosphate. These results indicate that Pyridoxal 5'-Phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.
Dorotea Natuzzi - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5′-Phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by Pyridoxal 5′-Phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5′-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC_50 of 0.5 m M , competed with the substrate for binding to the oxoglutaratecarrier ( K _ i = 0.4 m M ). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal5′-Phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [^3H]Pyridoxal 5′-Phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by Pyridoxal 5′-Phosphate. These results indicate that Pyridoxal 5′-Phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.
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Inactivation of the reconstituted oxoglutarate carrier from bovine heart mitochondria by Pyridoxal 5'-Phosphate.
Journal of bioenergetics and biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5'-Phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by Pyridoxal 5'-Phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal 5'-Phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]Pyridoxal 5'-Phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by Pyridoxal 5'-Phosphate. These results indicate that Pyridoxal 5'-Phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.
Soo Young Choi - One of the best experts on this subject based on the ideXlab platform.
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Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with Pyridoxal-5′-Phosphate
Journal of neurochemistry, 2008Co-Authors: Soo Young Choi, Jae Hoon Bahn, Byung Ryong Lee, Seong Gyu Jeon, Joong Sik Jang, Choong Kwon Kim, Li Hua Jin, Kyunghee Kim, Jin Sun Park, Jinseu ParkAbstract:An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by Pyridoxal-5'- Phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the Pyridoxal-5'-Phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by Pyridoxal-5'-Phosphate. The absorption spectrum of the reduced and dialyzed Pyridoxal-5'-Phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with Pyridoxal-5'-Phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of Pyridoxal-5'-Phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.
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Modulation of the Catalytic Activity of Brain Succinic Semialdehyde Reductase by Reaction with Pyridoxal 5′‐Phosphate
European journal of biochemistry, 1997Co-Authors: Joung-woo Hong, Sung-woo Cho, Jeong-suk Yoo, Byung Kwon Yoo, Kil Soo Lee, Soo Young ChoiAbstract:An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by Pyridoxal 5′-Phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by Pyridoxal 5′-Phosphate. After tryptic digestion of the enzyme modified with Pyridoxal 5′-Phosphate in the presence and absence of NADPH followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the Pyridoxal-5′-Phosphate–binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of Pyridoxal 5′-Phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.
Valentina Stipani - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5′-Phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by Pyridoxal 5′-Phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5′-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC_50 of 0.5 m M , competed with the substrate for binding to the oxoglutaratecarrier ( K _ i = 0.4 m M ). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal5′-Phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [^3H]Pyridoxal 5′-Phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by Pyridoxal 5′-Phosphate. These results indicate that Pyridoxal 5′-Phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.
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Inactivation of the reconstituted oxoglutarate carrier from bovine heart mitochondria by Pyridoxal 5'-Phosphate.
Journal of bioenergetics and biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5'-Phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by Pyridoxal 5'-Phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal 5'-Phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]Pyridoxal 5'-Phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by Pyridoxal 5'-Phosphate. These results indicate that Pyridoxal 5'-Phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.
Daniela V. Miniero - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5′-Phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by Pyridoxal 5′-Phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5′-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC_50 of 0.5 m M , competed with the substrate for binding to the oxoglutaratecarrier ( K _ i = 0.4 m M ). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal5′-Phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [^3H]Pyridoxal 5′-Phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by Pyridoxal 5′-Phosphate. These results indicate that Pyridoxal 5′-Phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.
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Inactivation of the reconstituted oxoglutarate carrier from bovine heart mitochondria by Pyridoxal 5'-Phosphate.
Journal of bioenergetics and biomembranes, 1999Co-Authors: Dorotea Natuzzi, Lucia Daddabbo, Valentina Stipani, Anna R. Cappello, Daniela V. Miniero, Loredana Capobianco, Italo StipaniAbstract:The effect of Pyridoxal 5'-Phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by Pyridoxal 5'-Phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-Phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by Pyridoxal 5'-Phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]Pyridoxal 5'-Phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by Pyridoxal 5'-Phosphate. These results indicate that Pyridoxal 5'-Phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.
