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Cytochrome b co-fractionates with gelatinase-containing granules in human neutrophils

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Summary

Subcellular fractionation studies in resting human neutrophils indicated a bimodal distribution for cytochrome b. A. major peak of cytochrome b co-sedimented with gelatinase under different experimental conditions. This localization was partially overlapped with specific granules (using lysozyme and lactoferrin as specific granule markers), but clearly resolved from azurophilic granules, plasma membrane, mitochondria, as well as from a novel alkaline phosphatase-rich intracellular organelle. A minor localization of cytochrome b was found in fractions enriched in both the plasma membrane marker 5′-nucleotidase and alkaline phosphatase. A significant portion of ubiquinone cell content co-fractionated with the gelatinase-containing granules. After phorbol myristate acetate (PMA)-cell stimulation, cytochrome b was mobilized to fractions showing respiratory burst activity and enriched in 5′-nucleotidase activity. This mobilization paralleled secretion of gelatinase and lysozyme to the extracellular medium. Furthermore, neutrophil stimulation with fluoride in the absence of cytochalasin B induced release of gelatinase and generation of superoxide anion with only minimal release of lysozyme. Preincubation of cells with the anion channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) prevented lysozyme release, but had only a minor effect on the release of gelatinase and did not inhibit the superoxide anion generation elicited by N-formyl-methionyl-leucyl-phenylalanine or PMA. These results suggest a main location of cytochrome b in mobilizable gelatinase-containing granules, which can constitute a subpopulation of specific granules. Furthermore, these findings show that the gelatinase-containing granule is functionally involved in the respiratory burst in neutrophils and that membrane fusion between plasma membrane and the gelatinase-containing granule occurs during activation of cells.

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Abbreviations

DIDS:

4,4′-diisothiocyanostilbene-2,2′-disulfonic acid

FMLP:

N-formyl-methionyl-leucyl-phenylalanine

PMA:

4β-phorbol, 12-myristate, 13-acetate

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Authors and Affiliations

  1. Centro de Investigaciones Biológicas, C. S. I. C., Velázquez 144, E-28006, Madrid, Spain

    Faustino Mollinedo & Consuelo Gajate

  2. Department of Biochemistry, Dartmouth Medical School, 03756, Hanover, New Hampshire, USA

    Donald L. Schneider

Authors
  1. Faustino Mollinedo
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  2. Consuelo Gajate
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  3. Donald L. Schneider
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Mollinedo, F., Gajate, C. & Schneider, D.L. Cytochrome b co-fractionates with gelatinase-containing granules in human neutrophils. Mol Cell Biochem 105, 49–60 (1991). https://doi.org/10.1007/BF00230374

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  • DOI: https://doi.org/10.1007/BF00230374

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