Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides of 91,000 and 22,000 relative molecular weights

Journal of Clinical Investigation (Impact Factor: 13.22). 10/1987; 80(3):732-42. DOI: 10.1172/JCI113128
Source: PubMed


A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.


Available from: Al Jesaitis, Dec 19, 2014
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    • "NADPH oxidase can produce hydrogen peroxide and superoxide which are needed in the bactericidal action of phagocytes.2 NADPH oxidase is composed of 2 plasma membrane subunits, gp91-phox and p22-phox, which comprise the cytochrome b558 complex.3,4,5,6 P47-phox, p67-phox, p40-phox and rac-2 are also important cytosolic oxidase components.3,4,5,6 "
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    ABSTRACT: Chronic granulomatous disease (CGD) is a rare immunodeficiency disease, which is characterized by the lack of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. The disease presents leukocytosis, anemia, hypergammaglobulinemia, and granuloma formation of the skin, lung, or lymph nodes. The mutation of the CYBB gene encoding gp91phox, located on chromosome Xp21.1 is one of the causes of CGD. We report a patient with X-linked CGD who carried a novel mutation, a c.1133A>G (paAsp378Gly) missense mutation, in the CYBB gene.
    Allergy, asthma & immunology research 07/2014; 6(4):366-9. DOI:10.4168/aair.2014.6.4.366 · 2.43 Impact Factor
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    • "This heterodimeric flavocytochrome is also called cytochrome b 558 , based on its spectroscopic properties. These subunits were named after their molecular mass on gel electrophoresis whereas the letters indicate a protein (p) or glycoprotein (gp) of the phagocyte oxidase (phox) (Dinauer et al., 1987; Parkos et al., 1987, 1988; Rotrosen et al., 1992). "
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    Pharmacological reviews 02/2014; 66(2):513-69. DOI:10.1124/pr.112.007351 · 17.10 Impact Factor
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    • "The best studied member of this group is the mammalian phagocytic gp91phox (named also Nox2), which is responsible for the defense response of neutrophils [7]. To activate its function a multi-subunit complex has to be formed: the integral membrane protein flavocytochrome b558, consisting of the catalytic subunit gp91phox and the adaptor protein p22phox [8], is assembled with the cytosolic components p40phox, p47phox, p67phox and the small GTPase Rac upon phosphorylation of p47phox [9]. NADPH oxidases have been found in a wide range of eukaryotic organisms, and have originally been linked with multicellularity [10]. "
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    ABSTRACT: NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ΔbcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination.
    PLoS ONE 06/2013; 8(2):e55879. DOI:10.1371/journal.pone.0055879 · 3.23 Impact Factor
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