The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.
Abstract: 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.
Publication Date: 1982-05-15 PubMed ID: 7115343PubMed Central: PMC1158375DOI: 10.1042/bj2040479Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research determined the absorption coefficient of cytochrome b-245 in different animals, its oxidation process, and its possible role in the respiratory burst of neutrophils. The study utilized neutrophils, which are a type of white blood cell, from various animal species. The researchers investigated the behavior of a particular molecule, cytochrome b-245, in these cells and suggested this molecule could be important in the immune response.
Experiment Setup and Procedures
- The research involved neutrophil cells from humans, cows, horses, and pigs, where it was discovered that all contained low-potential cytochrome b.
- The isolated membranes were incubated anaerobically with either types of NADH or NADPH to investigate whether the cytochrome b underwent a reduction. Furthermore, the researchers used phorbol myristate acetate to stimulate the cells and observe any effect on the rate and extent of cytochrome b reduction.
- Similarly, cytochrome b was also found in a specific granule fraction of human neutrophils. The researchers determined its reduction potential which was -248 mV, very close to that of the plasma-membrane cytochrome b.
Results and Findings
- The experiment determined that the cytochrome b-245 molecule was present both in the plasma membrane and certain specific granules in human neutrophils.
- The oxidation of the reduced cytochrome b-245 was swift enough to suggest that it could be the oxidase in the respiratory burst of neutrophils. This is a process in which the white blood cells rapidly release reactive oxygen species to combat infections.
- They also found that cytochrome b of human neutrophils was able to form a complex with CO (carbon monoxide). The percentage of cytochrome that formed a complex varied, with approximately 40% at room temperature and 60% at a colder temperature. The concentration of CO that produced 50% binding was found to be 1.18 mM.
Conclusions
- The research suggests a connection between the oxidation of cytochrome b-245 and the respiratory burst in neutrophils. This is an interesting discovery as the respiratory burst is a significant part of the immune response. Therefore, the findings may have important implications for understanding and treating diseases where the immune response is important.
Cite This Article
APA
Cross AR, Higson FK, Jones OT, Harper AM, Segal AW.
(1982).
The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.
Biochem J, 204(2), 479-485.
https://doi.org/10.1042/bj2040479 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Carbon Monoxide / metabolism
- Cattle
- Cell Membrane / metabolism
- Cytochrome b Group
- Cytochromes / blood
- Horses
- Humans
- Kinetics
- NADP / metabolism
- Neutrophils / drug effects
- Neutrophils / metabolism
- Oxidation-Reduction
- Potentiometry
- Spectrophotometry
- Swine
- Tetradecanoylphorbol Acetate / pharmacology
References
This article includes 14 references
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- Cross AR, Jones OT, Harper AM, Segal AW. Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst.. Biochem J 1981 Feb 15;194(2):599-606.
- Segal AW, Garcia R, Goldstone H, Cross AR, Jones OT. Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils.. Biochem J 1981 Apr 15;196(1):363-7.
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