Examination of the oxidase function of the b-type cytochrome in human polymorphonuclear leucocytes.
The spectral properties of a particulate fraction of human polymorphonuclear neutrophils capable of oxidizing NADPH were studied before and after depletion of myeloperoxidase by KCl treatment. Difference spectra (dithionite reduced minus oxidized) at 77 K of non-extracted particles showed peaks of a b-type cytochrome at 556, 527 and 425 nm and of myeloperoxidase at 636 and 474 nm. Extraction of myeloperoxidase led to a 4-5-fold increase in the size of the cytochrome b peaks. In non-extracted particles, the CO-reduced spectra at 77 K revealed a typical CO-reduced myeloperoxidase complex with new peaks at 625-630 and 462 nm, and a limited shift of the Soret band of reduced cytochrome b from 425 to 424-423 nm. The same shift was observed for cytochrome b in extracted particles. Photoirradiation of the CO-dithionite-reduced particles resulted in a back shift of the CO-reduced peaks to their original positions in the reduced spectrum. Concomitantly, the size of the peaks both for myeloperoxidase and cytochrome b was increased, indicating photoreduction. Cytochrome b and myeloperoxidase in neutrophil particles were poorly reduced by NADPH; reduction occurred upon photoirradiation. FAD and FMN added to particles in the presence of NADPH were photoreduced concomitantly with cytochrome b. Addition of phorbol myristate acetate to intact neutrophils in the presence of glucose resulted in CO- and cyanide-insensitive respiration, accumulation of O-2, and also in reduction of cytochrome b. The lag required to reach the steady-state production of O-2 was equal to the lag required for cytochrome b to reach a plateau of reduction. The data are consistent with the idea that cytochrome b in neutrophils might belong to a branched pathway that is not rate-limiting in the cyanide-resistant respiration of the neutrophils.
D002248:Carbon Monoxide / Q000494:pharmacology; D003573:Cytochrome b Group / Q000097:blood*; D004227:Dithionite / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008027:Light; D009249:NADP / Q000097:blood*; D009504:Neutrophils / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000097:blood*; D010101:Oxygen Consumption; D009195:Peroxidase / Q000097:blood; D011189:Potassium Chloride / Q000494:pharmacology
D003573:Cytochrome b Group; D004227:Dithionite; D009249:NADP; D011189:Potassium Chloride; D002248:Carbon Monoxide; D010088:Oxidoreductases; D009195:Peroxidase