title: Two pathways of activation of the superoxide-generating NADPH oxidase of phagocytes in vitro--distinctive effects of inhibitors.
abstract: The NADPH oxidase complex of phagocytes comprises a membrane-associated flavocytochrome b559, and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase Rac. Activation of the oxidase in vivo is the result of assembly of the cytosolic components with cytochrome b559 and is mimicked in vitro by a cell-free system consisting of membranes, p47phox, p67phox, nonprenylated or prenylated Rac, and an anionic amphiphile as activator (defined as "p47phox and amphiphile-dependent" or canonical pathway). We reported that prenylated Rac1 is capable of activating the NADPH oxidase in vitro in the absence of p47phox and amphiphile (defined as "p47phox and amphiphile-independent" pathway). We now demonstrate that the 2 pathways exhibit distinctive susceptibilities to inhibitors: 1) The anionic amphiphile lithium dodecyl sulfate, an activator of the canonical pathway, has the opposite effect (inhibition) on oxidase activation by prenylated Rac and p67phox; 2) GDP and, paradoxically, GTP (but not GMP, ATP, ADP, and AMP) prevent oxidase activation by the p47phox and amphiphile-independent pathway but do not affect activation by the canonical pathway; 3) The Rac-binding domain of p21-activated kinase is a potent inhibitor of activation by the p47phox and amphiphile-independent pathway while exerting a milder inhibitory effect on the canonical pathway; 4) The C-terminal polybasic Rac1 peptide 177-191 and the cationic antibiotic neomycin sulfate inhibit activation by the canonical pathway but do not affect activation by the p47phox and amphiphile-independent pathway; 5) Binding of prenylated Rac1 to membrane-mimicking phospholipid vesicles is, nevertheless, enhanced when these contain negatively charged lipids. It is proposed that preferential inhibition of oxidase activation, via the p47phox and amphiphile-independent pathway, is a reflection of interference by the inhibitors with Rac-dependent recruitment of p67phox to the membrane.
authors.affiliation: Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Natalia
authors.initials: N
authors.lastname: Sigal
authors.forename: Yara
authors.initials: Y
authors.lastname: Gorzalczany
authors.forename: Edgar
authors.initials: E
authors.lastname: Pick
mesh_terms: D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006168:Guinea Pigs; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010586:Phagocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D013481:Superoxides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1