A New Cytochrome in Neutrophilic Granules of Rabbit Leucocyte

Shinagawa, Yoshiya; Tanaka, Chikako; Teraoka, Akira; Shinagawa, Y.

doi:10.1093/oxfordjournals.jbchem.a128352

Cited by 54 publications

(9 citation statements)

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“…Neutrophils are known to contain a low-potential cytochrome (cytochrome b559) that appears to play an important, though as yet incompletely defined, role in O2 production by these cells (15,(26)(27)(28)(29)(30)(31)(32). Fig.…”

Section: Methodsmentioning

confidence: 99%

Respiratory burst oxidase from human neutrophils: purification and some properties.

Markert¹,

Glass²,

Babior³

1985

Proc. Natl. Acad. Sci. U.S.A.

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The respiratory burst oxidase of human neutrophils was purified by "dye-affinity" chromatography over a red agarose column. Electrophoresis of the purified enzyme on NaDodSO4 gel showed a single major band at 64,000-66,000 daltons, together with some minor contaminants. On a nondenaturing gel, the enzyme ran as two closely spaced bands, the faster of which contained flavin. When these two bands were rerun separately on a NaDodSO4 gel, they gave identical patterns, each showing a major band at ca. 65,00Q daltons. The specific activity (mean ± SEM) of the purified enzyme was 8.8 ± 3.5 ,umol of O2 per min/mg of protein.When exposed to appropriate stimuli, neutrophils are induced to express the "respiratory burst," a profound alteration in oxygen metabolism whose purpose is the generation of microbicidal oxidants by the (12)(13)(14)(15)(16).The respiratory burst oxidase was first solubilized from the plasma membranes of activated human neutrophils in 1978 (17). We now report the purification of this oxidase and describe some of the properties of the purified enzyme. MATERIALS AND METHODSHuman neutrophils were prepared by dextran sedimentation and differential centrifugation as described (18). Zymosan (Sigma) was boiled for 10 min in 1 M NaOH, then washed three times with Hanks' balanced salt solution (GIBCO), and finally opsonized as described elsewhere (3). Superoxide dismutase, horse heart cytochrome c (type VI), phosphatidylethanolamine (ovine brain), Lubrol PX, sodium deoxycholate, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, FAD, and NADPH were purchased from Sigma. The sodium deoxycholate was purified by recrystallization from absolute ethanol. Red Sepharose CL-6B (agarose coupled to reactive red 120) was obtained from Pharmacia. The concentration of dye in the lots purchased from Pharmacia ranged from 2.0 to 3.0 Afmol/ml of packed gel. Other reagents were the best grade commercially available apd were used without further purification.Gel Electrophoresis. Electrophoresis on 7.5% nondenaturing slab gels was performed by a modification of the method of Davis (19) in which the solution for preparing the lower gel contained 4% glycerol plus twice the usual concentration of Tris, that for preparing the upper gel contained half the usual concentration of bisacrylamide, and polymerization was accomplished with 0.035% (wt/vol) ammonium persulfate, which was removed by preelectrophoresis for 1 hr before the samples were applied to the slab. Nondenaturing gel electrophoresis was carried out at 40C. NaDodSO4/polyacrylamide gel electrophoresis (NaDodSO4/PAGE) was performed by the method of Laemmli (20) using 9o slab gels. Where necessary, samples were concentrated before electrophoresis in CF 25A ultrafiltration cones (Amicon) and then centrifuged for 30 min at 100,000 x g to remove aggregated proteins. Proteins were visualized with Coomassie blue R-250 or with a silver stain kit purchased from Bio-Rad.Localization of Flavin in the Nondenaturing Gel. The location of flavin in the nondenaturing gel was ...

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Section: Methodsmentioning

confidence: 99%

Respiratory burst oxidase from human neutrophils: purification and some properties.

Markert¹,

Glass²,

Babior³

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…This reaction, which produces a blue compound, indophenolblue, from dimethyl para -phenylen diamine and α-napthol [146], was originally thought to be produced by a specific enzyme, indophenol oxidase, but later shown to be a nonspecific indicator of oxidases. Subsequently it was also shown to be associated with rabbit neutrophils, localising to the granules [278]. In a thorough chromatographic characterisation of this cytochrome, the possibility was even raised that this new cytochrome might have something to do with neutrophil respiration insensitive to cyanide and antimycin A [277].…”

Section: Respiratory Burst—historymentioning

confidence: 99%

“…Subsequently it was also shown to be associated with rabbit neutrophils, localising to the granules [278]. In a thorough chromatographic characterisation of this cytochrome, the possibility was even raised that this new cytochrome might have something to do with neutrophil respiration insensitive to cyanide and antimycin A [277]. This linkage was never clearly made, possibly because it was thought the cytochrome might be an unusual cytochrome P420 (an inactivated form of cytochrome P450), and these reports in the Japanese literature were overlooked [169,170].…”

Section: Respiratory Burst—historymentioning

confidence: 99%

The NADPH oxidase of professional phagocytes—prototype of the NOX electron transport chain systems

Cross

Segal

2004

Biochimica et Biophysica Acta (BBA) - Bioenergetics

415

328

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The NADPH oxidase is an electron transport chain in "professional" phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.

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“…First, elucidation of the nature and composition of the phagocyte oxidase followed from Babior et al's report. Extending earlier published observations that neutrophils from rabbits and horses possess an unusual b-type cytochrome that could operate in the phagocyte oxidase (18,19), Segal et al (20) demonstrated that membranes from normal human neutrophils contain cytochrome b 245 , which is absent from patients with X-linked CGD. In a series of elegant studies between 1978 and 1987, Segal provided evidence that cytochrome b 245 serves as the electron transferase in the superoxide-generating system of phagocytes, a hypothesis that provoked significant controversy (e.g., see Refs.…”

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confidence: 70%

Identification and Quantitation of Superoxide Anion: Essential Steps in Elucidation of the Phagocyte “Respiratory Burst”

Nauseef

2014

The Journal of Immunology

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A New Cytochrome in Neutrophilic Granules of Rabbit Leucocyte

Cited by 54 publications

References 0 publications

Respiratory burst oxidase from human neutrophils: purification and some properties.

Respiratory burst oxidase from human neutrophils: purification and some properties.

The NADPH oxidase of professional phagocytes—prototype of the NOX electron transport chain systems

Identification and Quantitation of Superoxide Anion: Essential Steps in Elucidation of the Phagocyte “Respiratory Burst”

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