Possible role of Rac-GTPase-activating protein in the termination of superoxide production in phagocytic cells

The mechanism leading to the termination of superoxide production of phagoc ytes is poorly understood. The aim of the present study was to investigate the involvement of the active (GTP-bound) form of the GTP-binding proteins in maintaining continuous electron transport through the reduced nicotinami de adenine dinucleotide phosphate (NADPH) oxidase complex. Activation of th e enzyme was carried out under in vitro conditions and a shift from the act ive to the inactive form of the GTP-binding protein was attained (i) by add ition of an excess of GDP to the assembled enzyme complex or (ii) by variat ion of the Rac-GTPase activating (Rac-GAP) capacity of the constituents of the cell-free system. Significant inhibition of O-2(.-) production was obse rved when guanine dinucleotides were added after the assembly of the active enzyme complex. The effect was specific for GDP and GDP beta S whereas ADP , CDP and UDP were ineffective. GTP was significantly less efficient in ind ucing superoxide production in a cell-free system containing endogenous GAP activity than in a system devoid of GAP activity. It is suggested that the active, GTP-bound form of Rac is required for sustained catalytic function and Rac-GAP proteins are involved in the downregulation of the oxidase. (C ) 1999 Elsevier Science Inc.