p21rac does not participate in the early interaction between p47-phox and cytochrome b558 that leads to phagocyte NADPH oxidase activation in vitro.
The phagocyte superoxide-generating NADPH oxidase, a multicomponent, membrane-bound electron transport chain, consists of cytochrome b558, p47-phox, p67-phox, and p21rac1 or p21rac2. The mechanisms of oxidase assembly are poorly understood. In previous studies using a cell-free NADPH oxidase system, we showed that preincubation of neutrophil membrane with neutrophil cytosol containing p47-phox, but not p67-phox, led to formation of a long-lived NADPH oxidase intermediate. This suggested that p47-phox interacted with cytochrome b558 in the early stages of oxidase assembly while p67-phox participated in a later stage. Peptides containing the sequence RGVHFIF (corresponding to amino acids 559-565 of the 91-kDa subunit of cytochrome b558) inhibit NADPH oxidase activity by blocking the early interaction between p47-phox and cytochrome b558. In the present study, we examined whether p21rac facilitated the interaction between p47-phox and cytochrome b558. We preincubated pure recombinant p47-phox with neutrophil membrane containing cytochrome b558 in the cell-free system. Superoxide-generating activity was subsequently reconstituted by adding pure rp67-phox and partially purified p21rac. RGVHFIF inhibited superoxide production if added to the cell-free system during preincubation of rp47-phox with membrane. RGVHFIF was markedly less inhibitory if added to the cell-free system after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox conferred no protection from inhibition of superoxide-generating activity by RGVHFIF added after preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)
Kleinberg
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ME
Research Service, Baltimore Veterans Affairs Medical Center, Maryland 21201.
Malech
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HL
Mital
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DA
Leto
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D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D016897:Respiratory Burst; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D009097:Multienzyme Complexes; D010455:Peptides; D010750:Phosphoproteins; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins