Evidence for a functional cytoplasmic domain of phagocyte oxidase cytochrome b558.
Cytoplasmic domains of transmembrane proteins play a critical role in cellular processes involving interactions between membrane and cytosolic components. Activation of the phagocytic cell respiratory burst oxidase, the electron transport chain responsible for superoxide anion (O2-.) production, requires membrane components including cytochrome b558 and several cytosolic proteins; but the biochemical interactions of these components are poorly understood. Cytochrome b558 is an electron transport component of the oxidase. A role for cytochrome b558 in the organization or integration of other oxidase components has also been hypothesized. Antibodies binding the cytoplasmic carboxyl-terminal tail of the transmembrane 91-kDa subunit of cytochrome b558 specifically inhibited an amphiphile-activated cell-free O2-.-generating system that requires neutrophil membranes and cytosol. Synthetic peptides encompassing a 7-amino acid carboxyl-terminal sequence (RGVHFIF) within the same region of the 91-kDa subunit blocked activation of the oxidase by arachidonate, but did not affect activity of the assembled oxidase when added after arachidonate to the cell-free O2-.-generating system. The same peptides inhibited activation of the respiratory burst when allowed to diffuse into electrically permeabilized neutrophils before stimulation with formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These studies define a functional cytoplasmic domain of the transmembrane 91-kDa subunit of cytochrome b558 which may mediate interactions with other cellular proteins essential to activation of the phagocyte respiratory burst.
Rotrosen
D
D
Bacterial Diseases Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Kleinberg
M E
ME
Nunoi
H
H
Leto
T
T
Gallin
J I
JI
Malech
H L
HL
D000595:Amino Acid Sequence; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000097:blood*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001095:Arachidonic Acids; D003573:Cytochrome b Group; D046911:Macromolecular Substances; D009842:Oligopeptides; D013481:Superoxides; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate