The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.
1. The absorption coefficient of human neutrophil plasma-membrane reduced-minus-oxidized cytochrome b-245 was determined [delta epsilon (mM; 559-540 nm) = 21.6 cm-1]. 2. Neutrophil polymorphonuclear leucocytes (neutrophils) were prepared from human, ox, horse and pig blood. In each case plasma-membrane fractions were found to contain low-potential cytochrome b. When membranes from horse neutrophils were incubated anaerobically with either NADH or NADPH the cytochrome b became reduced. Prior stimulation of the cells with phorbol myristate acetate did not increase the rate or extent of cytochrome b reduction in isolated membranes, but did increase both the rate and extent of reduction by NADPH in Triton-treated cells. 3. A cytochrome b was present also in the specific granule fraction of human neutrophils. Its Em (pH 7.0) was found to be -248 mV, very similar to that of the plasma-membrane cytochrome b. 4. The rate of oxidation of reduce cytochrome b-245 by air-saturated buffer, was determined by using stopped-flow techniques. In intact membranes t 1/2 for oxidation was 4.7 ms. This rate is sufficiently rapid to support the view that cytochrome b-245 is the oxidase in the respiratory burst of neutrophils. 5. Plasma-membrane cytochrome b of human neutrophils formed a complex with CO. At room temperature and 1 atm of CO approx. 40% of the cytochrome formed a complex; approx. 60% binding was measured at the increased concentration of dissolved CO achieved at 5 degrees C. The concentration of CO giving 50% binding was 1.18 mM.
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000097:blood*; D006736:Horses; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D011199:Potentiometry; D013053:Spectrophotometry; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003573:Cytochrome b Group; D003580:Cytochromes; C035130:cytochrome b245; D009249:NADP; D002248:Carbon Monoxide; D013755:Tetradecanoylphorbol Acetate