GTPase activity of small GTP‐binding proteins in HL‐60 membranes is stimulated by arachidonic acid

Abstract

The GTPase activity of membranes isolated from differentiated HL-60 cells was investigated to obtain information about the possible involvement of membrane-bound GTP-binding proteins in the regulation of the NADPH oxidase. A more than tenfold increase in the rate of hydrolysis of membrane-bound GTP was observed when cytosol and arachidonic acid were added simultanously, i.e. under the same conditions where NADPH oxidase becomes activated. There were parallel changes in GTPase and NADPH oxidase activities when the concentration of arachidonic acid or the species of the fatty acid was varied or different detergents were applied. Separation of the GTP-binding proteins of the solubilized membrane by sucrose density gradient centrifugation, allowed us to ascribe the observed effect to the stimulation of the GTPase activity of small GTP-binding proteins by cytosolic component(s). Indirect evidence suggests that, in contrast to the effect upon recombinant ras and ras-GTPase-activating protein, in intact HL-60 membranes the interaction of rapl A with rap-GTPase-activating protein, is strongly enhanced by arachidonic acid.In resting phagocytosing cells, the superoxide (0, -)-producing NADPH oxidase is present in a dormant state. Following various stimulations, the active enzyme is rapidly assembled from the membrane-bound component cytochrome b,,, and two proteins of cytosolic origin, p47,,,,, and ~6 7 ,~, , [l, 21. Association of p47,,,, and p67,,, with the plasma membrane has been demonstrated both in intact cells [3-51 and in the in-vitru-activation system [6, 71. Using in vitro conditions, the prerequisite for the translocation is the presence of a suitable amphiphilic molecule, such as SDS or arachidonic acid [8-lo].Activation of the NADPH oxidase has been shown to be enhanced by GTP and poorly hydrolyzable GTP analogs and to be inhibited by GDP and its poorly hydrolyzable analog [ll-131. These findings suggested the participation of a GTP-binding protein in the process. Convincing evidence has been published recently by three different groups [14-171 that rac, a small GTP-binding protein, is the third cytosolic factor necessary for the activation of the oxidase and that it is responsible for the GTP dependence of the process. These results have been elegantly supported by the successful reconstitution of the fully active NADPH oxidase enzyme from purified cytochrome b,,, and recombinant p47ph,,, ~6 7~, , , , and racl [19].Another small GTP-binding protein, rapl A, has been reported to be firmly associated to cytochrome b,,, both in the plasma membrane and the specific granule membranes. TheCorrespondence to E. Ligeti two proteins could be co-immunopurified with antibodies against either of them [20, 211. Phosphorylation of raplA by CAMP-dependent protein kinase was shown to prevent its specific association with cytochrome b,,, [22]. Earlier experiments indicated that stimuli resulting in an increase in the intracellular CAMP concentration inhibit 0; -production [23, 241. All these data indicate a possible involvem...

Arachidonic Acid Activates the Noncapacitative Entry of Ca2+ during [Ca2+] Oscillations

“…In this context it is interesting that it has been suggested that capacitative activation of Ca 2ϩ entry may involve a GTP-dependent step requiring the hydrolysis of GTP to GDP such as might be mediated by a so-called small G protein (38,39). The reported participation of arachidonic acid in the regulation of such small G proteins (15,40) may indicate that they provide the common link for both capacitative and noncapacitative mechanisms of agonist-induced activation of Ca 2ϩ entry.…”

Phospholipids Can Switch the GTPase Substrate Preference of a GTPase-activating Protein

“…Alternatively, where noted, the amount of free [ 32 P]phosphate following GTP hydrolysis was determined after separation from [␥-32 P]GTP by active charcoal (14). In Fig.…”

Arachidonic acid in the reticuloendothelial network

“…Arachidonic acid activates NADPH oxidase [7,26]. This arachidonic acid release may be obligatory in Fc y receptor stimulation [ 51, and subsequent G-protein activation [8]. Arachidonic acid may stimulate the activity of NADPH oxidase by increasing the number of its active forms and its affinity for its substrate.…”