title: The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides.
abstract: NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.
authors.affiliation: Institut für Pharmakologie, Freie Universität Berlin.
authors.forename: R
authors.initials: R
authors.lastname: Seifert
authors.forename: W
authors.initials: W
authors.lastname: Rosenthal
authors.forename: G
authors.initials: G
authors.lastname: Schultz
authors.forename: T
authors.initials: T
authors.lastname: Wieland
authors.forename: P
authors.initials: P
authors.lastname: Gierschick
authors.forename: K H
authors.initials: KH
authors.lastname: Jakobs
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D010770:Phosphotransferases / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D000227:Adenine Nucleotides; D001095:Arachidonic Acids; D006150:Guanine Nucleotides; D013873:Thionucleotides; D013481:Superoxides; D006153:Guanosine Diphosphate; D016718:Arachidonic Acid; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D010770:Phosphotransferases; C021892:nucleoside phosphotransferase; D019204:GTP-Binding Proteins