The assembly of neutrophil NADPH oxidase: effects of mastoparan and its synthetic analogues.
Detergent-mediated activation of the phagocyte superoxide-generating NADPH oxidase requires the participation of at least four proteins: the membrane-bound heterodimeric cytochrome b558 and three cytosolic components, p47-phox, p67-phox and a Rac1/Rac2 protein. Peptides corresponding to sequences of different subunits of NADPH oxidase have been used as probes of the mechanism and sequence of assembly of the active complex. In the present study effects of mastoparans on activation of NADPH oxidase were investigated. Mastoparans are wasp venom cationic amphiphilic tetradecapeptides capable of modulation of various cellular activities. Natural mastoparans, as well as several synthetic mastoparan analogues, unrelated to oxidase components, blocked activation of the oxidase in the cell-free system (EC50 = 1.5 microM) and in guanosine 5'-[gamma-thio]triphosphate (GTP[S])/ATP-stimulated neutrophils permeabilized with streptolysin O. In the cell-free system the effect was not relieved by raising the detergent concentration and could not be ascribed to changes in critical micellar concentration values of the activating SDS or arachidonate. Chromatography of neutrophil cytosol on an immobilized mastoparan column suggested interaction of cytosolic p47-phox and p67-phox with the peptide. In spite of this interaction mastoparan did not interfere with translocation of p47-phox and p67-phox to the cell membranes.
Tisch
D
D
Department of Biochemistry, Tel-Aviv University, Israel.
Sharoni
Y
Y
Danilenko
M
M
Aviram
I
I
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002463:Cell Membrane Permeability; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000097:blood; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000097:blood*; D009252:NADPH Dehydrogenase / Q000097:blood; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D010750:Phosphoproteins / Q000097:blood; D011499:Protein Processing, Post-Translational; D013301:Streptolysins; D013329:Structure-Activity Relationship; D014862:Wasp Venoms / Q000737:chemistry / Q000494:pharmacology*
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D010455:Peptides; D010750:Phosphoproteins; D013301:Streptolysins; D014862:Wasp Venoms; C079789:neutrophil cytosol factor 67K; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase