In vitro Activation of the NADPH Oxidase by Fluoride

Abstract

The possible mechanism of activation of the NADPH oxidase by fluoride was investigated in the cell-free system. It is shown that the stirnulatory effect of fluoride is inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP [S]) and potentiated by GTP. The effect of fluoride is not additive with GTP [S].Fluoride activation requires the presence of Mg2+ in millimolar concentration but is independent of Al". The activating effect of fluoride is preserved in solubilized membrane extract after removal of the majority of heterotrimeric GTP-binding proteins by immunoadsorption. Fluoride has no direct action either on the nucleotide exchange or GTP hydrolysis of the isolated Rac protein. In contrast, fluoride effectively inhibits Rac-GTPase activity enhanced by a membrane component. In this way, fluoride could prolong the prevalence of Rac in the GTP-bound state and, as a consequence, activate NADPH oxidase. The possibility of the involvement of a membrane-bound Rac GTPase-activating protein activity in the physiological regulation of the enzyme is raised.

Regulation and termination of NADPH oxidase activity

“…Using crude membrane and cytosol preparations, the rate of O 2 .-production achieved in the presence of GTP is only 20-30 % of the maximal rate obtained in the presence of a non-hydrolyzable GTP analog such as GTPgS ( figure 6). However, application of fluoride, an inhibitor of GAPs [163][164][165], results in a concentration-dependent increase in the rate of GTP-elicited O 2 .-production approaching the values obtained with GTPgS ( fig. 6).…”

Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils

“…NaF was previously shown to stabilize the smg-GAP complex [59]. In our earlier experiments, addition of NaF reduced the RacGAP effect on O2 •--production of neutrophil membranes [36][37][38]40], but it did not affect the activity of purified cytochrome b558 [38,40]. These findings suggested that NaF acted by sequestering the GAP(s) -present in neutrophil membranes but absent in the cytochrome b558 preparation -into a stable complex.…”

“…Due to their complex domain structure [32,34] and extensive regulation [30,35], RacGAPs could fine tune Nox activity. In fact, a role for RacGAPs in the regulation of assembly and function of the Nox2 complex has been indicated in previous in vitro studies [36][37][38][39][40] and in genetically modified animals [41][42][43]. These were all isolated studies and comparative aspects have not been raised.…”

Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase

“…The third group of proteins are GTPase-activating proteins (GAPs), which stimulate the intrinsic rate of hydrolysis and thus accelerate the inactivation of the protein. We have previously demonstrated that fluoride ions inhibit a membrane-bound Rac-GAP activity in the crude membrane fraction of neutrophil granulocytes [28]. We proposed that the stimulatory effect of fluoride on NADPH oxidase under cell-free conditions reported by several groups [29][30][31][32] might be explained by the down-regulation of Rac-GAP activity in the system.…”