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Shozo Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a Purified Enzyme.
Biochimica et biophysica acta, 1999Co-Authors: Kazuhisa Katayama, Natsuo Ueda, Itsuo Katoh, Shozo YamamotoAbstract:Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat Enzyme with a hexahistidine tag in a baculovirus-insect cell expression system, and Purified the Enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 micromol/min/mg protein. The Purified recombinant Enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the Purified Enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4x10(-3) (37 degrees C, pH 9.0). These experimental results with a Purified Enzyme preparation quantitatively confirmed the reversibility of the Enzyme reaction previously observed with crude Enzyme preparations.
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Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a Purified Enzyme.
Biochimica et Biophysica Acta, 1999Co-Authors: Kazuhisa Katayama, Natsuo Ueda, Itsuo Katoh, Shozo YamamotoAbstract:Abstract Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat Enzyme with a hexahistidine tag in a baculovirus–insect cell expression system, and Purified the Enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 μmol/min/mg protein. The Purified recombinant Enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the Purified Enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4×10−3 (37°C, pH 9.0). These experimental results with a Purified Enzyme preparation quantitatively confirmed the reversibility of the Enzyme reaction previously observed with crude Enzyme preparations.
T K Harden - One of the best experts on this subject based on the ideXlab platform.
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A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the Purified Enzyme reconstituted with turkey erythrocyte ghosts.
The Journal of biological chemistry, 1990Co-Authors: Andrew J. Morris, G L Waldo, C P Downes, T K HardenAbstract:The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the Purified turkey erythrocyte phospholipase C after reconstitution of the Enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of Purified Enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of Purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of Purified Enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-Purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the Purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the Purified Enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.
Kazuhisa Katayama - One of the best experts on this subject based on the ideXlab platform.
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Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a Purified Enzyme.
Biochimica et biophysica acta, 1999Co-Authors: Kazuhisa Katayama, Natsuo Ueda, Itsuo Katoh, Shozo YamamotoAbstract:Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat Enzyme with a hexahistidine tag in a baculovirus-insect cell expression system, and Purified the Enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 micromol/min/mg protein. The Purified recombinant Enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the Purified Enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4x10(-3) (37 degrees C, pH 9.0). These experimental results with a Purified Enzyme preparation quantitatively confirmed the reversibility of the Enzyme reaction previously observed with crude Enzyme preparations.
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Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a Purified Enzyme.
Biochimica et Biophysica Acta, 1999Co-Authors: Kazuhisa Katayama, Natsuo Ueda, Itsuo Katoh, Shozo YamamotoAbstract:Abstract Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat Enzyme with a hexahistidine tag in a baculovirus–insect cell expression system, and Purified the Enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 μmol/min/mg protein. The Purified recombinant Enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the Purified Enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4×10−3 (37°C, pH 9.0). These experimental results with a Purified Enzyme preparation quantitatively confirmed the reversibility of the Enzyme reaction previously observed with crude Enzyme preparations.
Takayuki Hoson - One of the best experts on this subject based on the ideXlab platform.
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a new type of endo xyloglucan transferase devoted to xyloglucan hydrolysis in the cell wall of azuki bean epicotyls
Plant and Cell Physiology, 2001Co-Authors: Akira Tabuchi, Hitoshi Mori, Seiichiro Kamisaka, Takayuki HosonAbstract:;A new type of xyloglucan-degrading Enzyme was isolated from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls and its characteristics were determined. The Enzyme was Purified to apparent homogeneity by Concanavalin A (Con A)-Sepharose, cation exchange, and gel filtration columns from a cell wall protein fraction extracted with 1 M sodium chloride. The Purified Enzyme gave a single protein band of 33 kDa on SDS-PAGE. The Enzyme specifically cleaved xyloglucans and showed maximum activity at pH 5.0 when assayed by the iodine-staining method. An increase in reducing power in xyloglucan solution was clearly detected after treatment with the Purified Enzyme. Xyloglucans with molecular masses of 500 and 25 kDa were gradually hydrolyzed to 5 kDa for 96 h without production of any oligo- or monosaccharide with the Purified Enzyme. The Purified Enzyme did not show an endo-type transglycosylation reaction, even in the presence of xyloglucan oligosaccharides. Partial amino acid sequences of the Enzyme shared an identity with endo-xyloglucan transferase (EXGT) family, especially with xyloglucan endotransglycosylase (XET) from nasturtium. These results suggest that the Enzyme is a new member of EXGT devoted solely to xyloglucan hydrolysis.
Andrew J. Morris - One of the best experts on this subject based on the ideXlab platform.
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A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the Purified Enzyme reconstituted with turkey erythrocyte ghosts.
The Journal of biological chemistry, 1990Co-Authors: Andrew J. Morris, G L Waldo, C P Downes, T K HardenAbstract:The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the Purified turkey erythrocyte phospholipase C after reconstitution of the Enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of Purified Enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of Purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of Purified Enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-Purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the Purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the Purified Enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.
