The Experts below are selected from a list of 57 Experts worldwide ranked by ideXlab platform
Maria A. Vanoni - One of the best experts on this subject based on the ideXlab platform.
-
quaternary structure of azospirillum brasilense nadph dependent glutamate synthase in solution as revealed by synchrotron radiation x ray scattering
Journal of Biological Chemistry, 2003Co-Authors: Maxim V Petoukhov, Bruno Curti, Sergio Ravasio, Dmitri I Svergun, Peter V Konarev, Robert H H Van Den Heuvel, Maria A. VanoniAbstract:Abstract Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent Enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS αβ holoenzyme and its isolated α and β subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS α subunit and αβ holoenzyme were found to be tetrameric in solution, whereas the β subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of α subunits in the (αβ)4 holoenzyme is similar to that in the tetrameric α4 complex and that β subunits occupy the periphery of the holoenzyme. The structure of α4 was further modeled using the available crystallographic coordinates of the monomeric α subunit assuming P222 symmetry. To model the entire αβ holoenzyme, a putative αβ protomer was constructed from the coordinates of the α subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the β subunit. Rigid body refinement yielded a model of GltS with an arrangement of α subunits similar to that in α4, but displaying contacts also between β subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the αβ protomers, which is consistent with the available biochemical evidence.
-
Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated α subunit
Biochemistry, 2002Co-Authors: Sergio Ravasio, Bruno Curti, Andrea Mattevi, Laura Dossena, Eugenio Martín-figueroa, Francisco J. Florencio, Paola Morandi, Maria A. VanoniAbstract:The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent Enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.
Sergio Ravasio - One of the best experts on this subject based on the ideXlab platform.
-
quaternary structure of azospirillum brasilense nadph dependent glutamate synthase in solution as revealed by synchrotron radiation x ray scattering
Journal of Biological Chemistry, 2003Co-Authors: Maxim V Petoukhov, Bruno Curti, Sergio Ravasio, Dmitri I Svergun, Peter V Konarev, Robert H H Van Den Heuvel, Maria A. VanoniAbstract:Abstract Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent Enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS αβ holoenzyme and its isolated α and β subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS α subunit and αβ holoenzyme were found to be tetrameric in solution, whereas the β subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of α subunits in the (αβ)4 holoenzyme is similar to that in the tetrameric α4 complex and that β subunits occupy the periphery of the holoenzyme. The structure of α4 was further modeled using the available crystallographic coordinates of the monomeric α subunit assuming P222 symmetry. To model the entire αβ holoenzyme, a putative αβ protomer was constructed from the coordinates of the α subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the β subunit. Rigid body refinement yielded a model of GltS with an arrangement of α subunits similar to that in α4, but displaying contacts also between β subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the αβ protomers, which is consistent with the available biochemical evidence.
-
Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated α subunit
Biochemistry, 2002Co-Authors: Sergio Ravasio, Bruno Curti, Andrea Mattevi, Laura Dossena, Eugenio Martín-figueroa, Francisco J. Florencio, Paola Morandi, Maria A. VanoniAbstract:The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent Enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.
Takami Takizawa - One of the best experts on this subject based on the ideXlab platform.
-
Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry.
European journal of histochemistry : EJH, 2009Co-Authors: Shigeki Matsubara, D. Matsubara, T Ishibashi, Takami TakizawaAbstract:Although various tissue macrophages possess high glucose6-phosphate dehydrogenase (G6PD) activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells) has not been determined. We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide) method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent Enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.
-
Enzyme-cytochemically Detectable Glucose-6-phosphate Dehydrogenase in Human Villous Macrophages (Hofbauer Cells)
Placenta, 2001Co-Authors: Shigeki Matsubara, T. Takayama, R. Iwasaki, Norio Komatsu, D. Matsubara, Takami Takizawa, Ikuo SatoAbstract:Though various tissue macrophages possess high glucose-6-phosphate dehydrogenase (G6PD) activity, which plays an important role in their phagocytosis/bactericidal function, the presence of this enzyme in human placental villous macrophages (Hofbauer cells) has not been determined. We examined the ultrastructural localization of glucose-6-phosphate dehydrogenase (G6PD) in Hofbauer cells in first and second trimester placental villi, using a newly developed enzyme-cytochemistry (copper-ferrocyanide) method. Electron-dense deposits indicative of G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Hofbauer cells. Positive and negative cytochemical controls ensured specific detection of enzyme activity. These observations indicated that Hofbauer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Hofbauer cell G6PD may play a role in placental defense, by supplying NADPH-dependent Enzymes (i.e. nitric oxide synthase or NADPH oxidase) with NADPH. This enzyme may also fuel Hofbauer cells with ribose 5-phosphate during their cell proliferation and cell division.
Shigeki Matsubara - One of the best experts on this subject based on the ideXlab platform.
-
Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry.
European journal of histochemistry : EJH, 2009Co-Authors: Shigeki Matsubara, D. Matsubara, T Ishibashi, Takami TakizawaAbstract:Although various tissue macrophages possess high glucose6-phosphate dehydrogenase (G6PD) activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells) has not been determined. We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide) method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent Enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.
-
Enzyme-cytochemically Detectable Glucose-6-phosphate Dehydrogenase in Human Villous Macrophages (Hofbauer Cells)
Placenta, 2001Co-Authors: Shigeki Matsubara, T. Takayama, R. Iwasaki, Norio Komatsu, D. Matsubara, Takami Takizawa, Ikuo SatoAbstract:Though various tissue macrophages possess high glucose-6-phosphate dehydrogenase (G6PD) activity, which plays an important role in their phagocytosis/bactericidal function, the presence of this enzyme in human placental villous macrophages (Hofbauer cells) has not been determined. We examined the ultrastructural localization of glucose-6-phosphate dehydrogenase (G6PD) in Hofbauer cells in first and second trimester placental villi, using a newly developed enzyme-cytochemistry (copper-ferrocyanide) method. Electron-dense deposits indicative of G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Hofbauer cells. Positive and negative cytochemical controls ensured specific detection of enzyme activity. These observations indicated that Hofbauer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Hofbauer cell G6PD may play a role in placental defense, by supplying NADPH-dependent Enzymes (i.e. nitric oxide synthase or NADPH oxidase) with NADPH. This enzyme may also fuel Hofbauer cells with ribose 5-phosphate during their cell proliferation and cell division.
Bruno Curti - One of the best experts on this subject based on the ideXlab platform.
-
quaternary structure of azospirillum brasilense nadph dependent glutamate synthase in solution as revealed by synchrotron radiation x ray scattering
Journal of Biological Chemistry, 2003Co-Authors: Maxim V Petoukhov, Bruno Curti, Sergio Ravasio, Dmitri I Svergun, Peter V Konarev, Robert H H Van Den Heuvel, Maria A. VanoniAbstract:Abstract Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent Enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS αβ holoenzyme and its isolated α and β subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS α subunit and αβ holoenzyme were found to be tetrameric in solution, whereas the β subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of α subunits in the (αβ)4 holoenzyme is similar to that in the tetrameric α4 complex and that β subunits occupy the periphery of the holoenzyme. The structure of α4 was further modeled using the available crystallographic coordinates of the monomeric α subunit assuming P222 symmetry. To model the entire αβ holoenzyme, a putative αβ protomer was constructed from the coordinates of the α subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the β subunit. Rigid body refinement yielded a model of GltS with an arrangement of α subunits similar to that in α4, but displaying contacts also between β subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the αβ protomers, which is consistent with the available biochemical evidence.
-
Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated α subunit
Biochemistry, 2002Co-Authors: Sergio Ravasio, Bruno Curti, Andrea Mattevi, Laura Dossena, Eugenio Martín-figueroa, Francisco J. Florencio, Paola Morandi, Maria A. VanoniAbstract:The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent Enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.
