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Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues. 1992

E Klinger, and I Aviram
Department of Biochemistry, Faculty of Life Sciences, Tel-Aviv University, Israel.

Activation of superoxide-producing NADPH oxidase of neutrophils requires the presence of cell membranes, cytosolic components and arachidonate and is markedly enhanced by non-hydrolysable analogues of guanine nucleotides, i.e. guanosine 5'-[gamma-thio]triphosphate and guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG). Gel filtration and ultrafiltration of the cytosol decreased the basal activity of NADPH oxidase. Activity could be restored by GTP, suggesting participation of the nucleotide in basal activation. Preincubation of neutrophil cytosol with periodate-oxidized p[NH]ppG (ox-p[NH]ppG) followed by gel filtration resulted in a time-dependent enhancement of basal oxidase activity. The presence of GDP or GTP, but not ATP, during the incubation with ox-p[NH]ppG abolished this enhancement. These data are consistent with a stable association of ox-p[NH]ppG with an oxidase-linked cytosolic protein. SDS/PAGE of neutrophil cytosol preincubated with [3H]ox-p[NH]ppG revealed radioactivity in bands migrating as 100, 70, 47, 34 and 22 kDa proteins. Evidence for covalent labelling of the cytosolic protein p47-phox with [3H]ox-p[NH]ppG is presented. Heterogeneity of cytosolic GTP-binding sites and possible participation of protein p47-phox in functional interaction with GTP analogues during cell-free activation are suggested.

UI MeSH Term Description Entries
D009247 NADH, NADPH Oxidoreductases A group of oxidoreductases that act on NADH or NADPH. In general, enzymes using NADH or NADPH to reduce a substrate are classified according to the reverse reaction, in which NAD+ or NADP+ is formally regarded as an acceptor. This subclass includes only those enzymes in which some other redox carrier is the acceptor. (Enzyme Nomenclature, 1992, p100) EC 1.6. Oxidoreductases, NADH, NADPH,NADPH Oxidoreductases NADH,Oxidoreductases NADH, NADPH
D009504 Neutrophils Granular leukocytes having a nucleus with three to five lobes connected by slender threads of chromatin, and cytoplasm containing fine inconspicuous granules and stainable by neutral dyes. LE Cells,Leukocytes, Polymorphonuclear,Polymorphonuclear Leukocytes,Polymorphonuclear Neutrophils,Neutrophil Band Cells,Band Cell, Neutrophil,Cell, LE,LE Cell,Leukocyte, Polymorphonuclear,Neutrophil,Neutrophil Band Cell,Neutrophil, Polymorphonuclear,Polymorphonuclear Leukocyte,Polymorphonuclear Neutrophil
D011233 Precipitin Tests Serologic tests in which a positive reaction manifested by visible CHEMICAL PRECIPITATION occurs when a soluble ANTIGEN reacts with its precipitins, i.e., ANTIBODIES that can form a precipitate. Precipitin Test,Test, Precipitin,Tests, Precipitin
D002474 Cell-Free System A fractionated cell extract that maintains a biological function. A subcellular fraction isolated by ultracentrifugation or other separation techniques must first be isolated so that a process can be studied free from all of the complex side reactions that occur in a cell. The cell-free system is therefore widely used in cell biology. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p166) Cellfree System,Cell Free System,Cell-Free Systems,Cellfree Systems,System, Cell-Free,System, Cellfree,Systems, Cell-Free,Systems, Cellfree
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D003600 Cytosol Intracellular fluid from the cytoplasm after removal of ORGANELLES and other insoluble cytoplasmic components. Cytosols
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004789 Enzyme Activation Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme. Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D006160 Guanosine Triphosphate Guanosine 5'-(tetrahydrogen triphosphate). A guanine nucleotide containing three phosphate groups esterified to the sugar moiety. GTP,Triphosphate, Guanosine
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man

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