Seifert, Roland and Rosenthal, W. and Schultz, Günter and Wieland, T. and Gierschick, P. and Jakobs, K. H.
The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides.
European journal of biochemistry / FEBS 175 (1), pp. 51-55.
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.
|Institutions:|| Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann formerly Prof. Seifert)|
|Leukemia, Myeloid, Acute/metabolism||MESH|
|NADH, NADPH Oxidoreductases/metabolism||MESH|
|Subjects:||600 Technology > 610 Medical sciences Medicine|
600 Technology > 615 Pharmacy
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||Unknown|
|Deposited On:||26 Jan 2012 08:34|
|Last Modified:||26 Jan 2012 08:34|