IN-VITRO ACTIVATION OF THE NADPH OXIDASE BY FLUORIDE - POSSIBLE INVOLVEMENT OF A FACTOR ACTIVATING GTP HYDROLYSIS ON PAC (PAC-GAP)

The possible mechanism of activation of the NADPH oxidase by fluoride was investigated in the cell-free system. It is shown that the stimula tory effect of fluoride is inhibited by guanosine 5'-O-(2-thiodiphosph ate) (GDP[S]) and potentiated by GTP. The effect of fluoride is not ad ditive with GTP[S]. Fluoride activation requires the presence of Mg2in millimolar concentration but is independent of Al3+. The activating effect of fluoride is preserved in solubilized membrane extract after removal of the majority of heterotrimeric GTP-binding proteins by imm unoadsorption. Fluoride has no direct action either on the nucleotide exchange or GTP hydrolysis of the isolated Rac protein. In contrast, f luoride effectively inhibits Rac-GTPase activity enhanced by a membran e 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-act ivating protein activity in the physiological regulation of the enzyme is raised.