title: Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues.
abstract: 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.
authors.affiliation: Department of Biochemistry, Faculty of Life Sciences, Tel-Aviv University, Israel.
authors.forename: E
authors.initials: E
authors.lastname: Klinger
authors.forename: I
authors.initials: I
authors.lastname: Aviram
mesh_terms: D015153:Blotting, Western; D002474:Cell-Free System; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D011233:Precipitin Tests
chemical_list: D006160:Guanosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases