The role of nucleoside‐diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides

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Abstract
NADPH‐oxidase‐catalyzed superoxide (O−2) 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′‐[γ‐thio]triphosphate (GTP[γS] and guanosine 5′‐[β,γ‐imido]triphosphate, being potent activators of guanine‐nucleotide‐binding proteins (G proteins), stimulated O−2 formation up to 3.5‐fold. The adenine analogue of GTP[γS], adenosine 5′‐[γ‐thio]triphosphate (ATP[γS]), which can serve as donor of thiophosphoryl groups in kinase‐mediated reactions, stimulated O−2 formation up to 2.5‐fold, whereas the non‐phosphorylating adenosine 5′‐[β,γ‐imido]triphosphate was inactive. The effect of ATP[γS] was half‐maximal at a concentration of 2 μM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[γS]. HL‐60 membranes exhibited nucleoside‐diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[γS] by ATP[γS]. GTP[γS] formation was half‐maximal at a concentration of 3–4 μM ATP[γS] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[γS] on O−2 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[γS] and considerably diminished stimulation by GTP[γS]. GTP[γS] also served as a thophosphoryl group donor to GDP, with an even higher efficiency than ATP[γS]. Transthiophosphorylation of GDP to GTP[γS] by GTP[γS] 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.