Activation of the O^–₂‐Generating NADPH Oxidase in a Semi–Recombinant Cell‐Free System

Abstract

The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotidebinding regulatory (G) protein Racl , were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Racl , the latter was not detected in the p47-p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Racl in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell-free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KC1-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.Phagocytes, among which neutrophils are the most studied, respond to soluble or particulate stimuli, by a large increase in oxygen consumption with concomitant production of the superoxide anion 0;. The enzyme complex which catalyzes the monoelectronic reduction of 0, to 0; is termed NADPH oxidase and is located in the plasma membrane. Oxidase activation corresponds to the functional assembly of a number of components including an heterodimeric redox protein of hydrophobic nature, the flavocytochrome b,,,, and water soluble proteins referred to as cytosolic factors, p47 and p67. The monomeric guanosine-nucleotide-binding regulatory (G) proteins, Rac and possibly RaplA, are also involved in the modulation of oxidase activation in a manner which is not yet clear (for review see Babior, 1992; Morel et al., 1991 ;Segal and Abo, 1993).The observation that addition of GTP to a cell-free system of oxidase activation significantly enhanced the elicited oxidase activity (Seifert and Schultz, 1987;Gabig et al., 1987;Ligeti et al., 1988) was the beginning of a series of investigations that ended with in vivo experiments in which Correspondence to A. Fuchs,

Autoinhibition of p50 Rho GTPase-activating Protein (GAP) Is Released by Prenylated Small GTPases

“…15 was used. Briefly, L40 yeast strain was transformed with the two plasmids using classical LiAc protocol.…”

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

“…GAP proteins were added either 10 min before or 10 min after AA. Membrane fractions isolated from human PMN contained sufficient Rac for NADPH oxidase assembly [38,49]. For each condition the optimal concentration of AA was determined and used in the activation mixture.…”

The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils

“…From the heme content, the amount of flavocytochrome b was deduced assuming the presence of two hemes per flavocytochrome b [17]. The recombinant cytosolic proteins, p47phox, p67phox and Rac2, prepared as described [18] were kindly provided by M. C. Dagher UMR 5092 CEA-Grenoble.…”