Studies of cytochrome b-245 translocation in the PMA stimulation of the human neutrophil NADPH-oxidase.
The human neutrophil respiratory burst, activated by phorbol 12-myristate 13-acetate (PMA), results from specific receptor-ligand binding and activation of the NADPH-oxidase in the plasma membrane. The role of granule membrane constituents has been elucidated with neutrophils disrupted by nitrogen cavitation and then fractionated in Percoll gradients to resolve four postnuclear fractions: cytoplasm, light membranes or gamma fraction (site of the NADPH-oxidase), a light granule (beta) fraction containing putative constituents of the NADPH-oxidase (cytochrome b-245 and an associated flavoprotein), and a fraction of heavy granules. Cytochrome b-245 is localized to two pools of specific granules within the beta fraction as assessed by differing sedimentation in narrow Percoll gradients and translocates upon PMA-stimulation from one of these specific granule sub-pools to the plasma membrane where it exhibits no change in its midpoint redox potential. Translocation of cytochrome b-245 parallels O2-production initiated by PMA stimulation as assessed in the time course of each activity. The finding of increased amounts of the b cytochrome in cytoplast membranes relative to plasma membranes of unstimulated cells suggests that the cytoplasts, devoid of granules yet capable of O2-generation upon PMA-stimulation, are useful in assessing post-translocation events in the activation pathway of the NADPH-oxidase. These data support the hypothesis that translocation of NADPH-oxidase components from an intracellular granular pool contributes to respiratory burst expression.
Higson
F K
FK
Durbin
L
L
Pavlotsky
N
N
Tauber
A I
AI
D002451:Cell Compartmentation / Q000187:drug effects; D003573:Cytochrome b Group / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010704:Phorbols / Q000494:pharmacology*; D013481:Superoxides / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D003573:Cytochrome b Group; D010704:Phorbols; C035130:cytochrome b245; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate