Direct evidence for interaction between COOH-terminal regions of cytochrome b558 subunits and cytosolic 47-kDa protein during activation of an O(2-)-generating system in neutrophils.
Cytochrome b558 in phagocytes is a transmembrane protein composed of large and small subunits and considered to play a key role in O2- generation during the respiratory burst. The COOH-terminal regions of the cytochrome subunits protrude to the cytoplasmic side and are assumed to be the sites for association with cytosolic components to form an active O(2-)-generating complex (Imajoh-Ohmi, S., Tokita, K., Ochiai, H., Nakamura, M., and Kanegasaki, S. (1992) J. Biol. Chem. 267, 180-184). We show here that two synthetic peptides corresponding to the COOH-terminal region of each subunit inhibit NADPH-dependent oxygen uptake induced by sodium dodecyl sulfate (SDS) in a cell-free system consisting of plasma membrane and cytosol. The inhibition was observed when either peptide was added to the system before, but not after, the activation with SDS suggesting that interaction between the COOH-terminal regions of the cytochrome subunits and cytosolic components is important for the assembly and the activity of the O(2-)-generating system. Using the cross-linking reagent dimethyl 3,3'-dithiobis-propionimidate, we found that the cytosolic 47-kDa protein, an essential component of the O(2-)-generating system, interacted with the synthetic peptides in the presence of SDS. In addition to the 47-kDa protein, a 17-kDa protein was found to be associated with the peptide corresponding to the COOH-terminal region of the small subunit. These results indicate that the cytosolic COOH-terminal regions of cytochrome b558 subunits are the binding sites for both the cytosolic 47-kDa protein and the 17-kDa protein and that the binding takes place during activation of the system.
Nakanishi
A
A
Institute of Medical Science, University of Tokyo, Japan.
Imajoh-Ohmi
S
S
Fujinawa
T
T
Kikuchi
H
H
Kanegasaki
S
S
D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000378:metabolism* / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism*
D003573:Cytochrome b Group; D046911:Macromolecular Substances; D010455:Peptides; D013481:Superoxides; C036888:cytochrome b558; D019255:NADPH Oxidases