scholarly journals The subcellular distribution and some properties of the cytochrome b component of the microbicidal oxidase system of human neutrophils

1979 ◽  
Vol 182 (1) ◽  
pp. 181-188 ◽  
Author(s):  
A W Segal ◽  
O T G Jones
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Concanavalin A ◽  
Subcellular Distribution ◽  
Human Neutrophils ◽  
Similar Distribution ◽  
Oxidase System ◽  
Dual Localization ◽  
Specific Granules ◽  
Analytical Fractionation

A novel cytochrome b has recently been described in human neutrophils. The subcellular distribution of this cytochrome was investigated by analytical fractionation on continuous sucrose gradients and it was found to have a dual localization, the major component having a similar distribution to the plasma-membrane marker [3H]concanavalin A, and a denser peak located with the specific granules. The two components were separated on discontinuous gradients and studied independently.

scholarly journals Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1563-1573 ◽  
Author(s):  
MT Quinn ◽  
ML Mullen ◽  
AJ Jesaitis ◽  
JG Linner
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Subcellular Distribution ◽  
Intact Cell ◽  
Human Neutrophils ◽  
Cytochrome B559 ◽  
Thin Sections ◽  
Specific Granule ◽  
Generating System ◽  
Peptide Antibodies

Abstract Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti- cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule- associated Rap1A was translocated to the plasma membrane in PMA- stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide- generating system.

scholarly journals Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1563-1573 ◽  
Author(s):  
MT Quinn ◽  
ML Mullen ◽  
AJ Jesaitis ◽  
JG Linner
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Subcellular Distribution ◽  
Intact Cell ◽  
Human Neutrophils ◽  
Cytochrome B559 ◽  
Thin Sections ◽  
Specific Granule ◽  
Generating System ◽  
Peptide Antibodies

Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti- cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule- associated Rap1A was translocated to the plasma membrane in PMA- stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide- generating system.

scholarly journals Isolation of plasma membrane from human neutrophils and determination of cytochrome b and quinone content

1981 ◽  
Vol 153 (5) ◽  
pp. 1316-1328 ◽  
Author(s):  
EP Sloan ◽  
DR Crawford ◽  
DL Schneider
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Protein A ◽  
Cell Protein ◽  
Human Neutrophils ◽  
Marker Enzyme ◽  
Whole Cell ◽  
Fold Enrichment ◽  
Dual Localization ◽  
Primary Location

Analyses of plasma membrane and other subcellular fractions indicate that the primary location of cytochrome b in human neutrophils is not the plasma membrane. The procedure developed for the purification of plasma membrane from fresh human neutrophils yielded a 14-fold enrichment in the marker enzyme 5'-nucleotidase and a 10-fold enrichment in ouabain-sensitive ATPase. On sucrose density gradients, the peak density of 5'-nucleotidase activity was 1.12 g/ml, and was shifted after digitonin addition to 1.15 g/ml. Protein in the plasma membrane equalled approximately 8 percent of the whole cell protein. A b-type cytochrome was found to be present in the plasma membrane fraction at a concentration of 205 pmol/mg of protein, which is three times greater than that in the neutrophil overall. Although this cytochrome has been reported previously in the neutrophil, this is the first determination for purified plasma membrane and may indicate that b-type cytochrome has a dual localization in the human neutrophil. Differential centrifugation results suggest that the primary location is in the granules, probably specific granules. Quinone content in the plasma membrane was found to be 740 pmol/mg of protein, a concentration two times greater than in the whole cell. Such a small enhancement of quinone indicates that quinone also is not primarily located in the plasma membrane.

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

scholarly journals Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils

1994 ◽  
Vol 299 (2) ◽  
pp. 473-479 ◽  
Author(s):  
H Sengeløv ◽  
F Boulay ◽  
L Kjeldsen ◽  
N Borregaard
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Beta Band ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Specific Granules ◽  
Inflammatory Stimuli

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

scholarly journals Changes in the subcellular distribution of the cytochrome b-245 on stimulation of human neutrophils

1984 ◽  
Vol 219 (1) ◽  
pp. 233-242 ◽  
Author(s):  
R C Garcia ◽  
A W Segal
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Bimodal Distribution ◽  
Human Neutrophils ◽  
Distribution Profile ◽  
Parallel Increase ◽  
Similar Density ◽  
Cytochrome D ◽  
Stimulation Of ◽  
Intracellular Structure

Cytochrome b-245 of neutrophils has a bimodal distribution in sucrose density gradients. The lighter component (d = 1.14) is shown to be associated with the plasma membrane by the similarity between its density and that of markers of this organelle, as well as a parallel increase in the density of the cytochrome and plasma membrane after treatment with digitonin or dimethyl suberimidate. The cytochrome b-245 of monocytes and cytoplasts, the latter produced by the removal of nuclei and granules from neutrophils, was located only in the plasma membrane. The denser peak of cytochrome (d = 1.19), which contained approximately half of the cytochrome b of neutrophils, had a similar density-distribution profile to the specific granules. After hypo-osmotic disruption of this denser material, the cytochrome distributed with the density of membranes, suggesting an original location within the membrane of the intracellular structure. Redistribution of the cytochrome from the granules to the membranes was observed after stimulation of respiratory activity with soluble agents or opsonized particles. This translocation is not responsible for activation of the oxidase system. There was poor agreement between the kinetics of the transfer of cytochromes from the dense component to the membranes, and degranulation of specific-granule contents, suggesting that the cytochrome may be located in another intracellular structure or that its localization becomes further modified after granule fusion.

scholarly journals Calcium-induced translocation of annexins to subcellular organelles of human neutrophils

1994 ◽  
Vol 300 (2) ◽  
pp. 325-330 ◽  
Author(s):  
C Sjölin ◽  
O Stendahl ◽  
C Dahlgren
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Consensus Sequence ◽  
Annexin Ii ◽  
Secretory Process ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Annexin I ◽  
Specific Granules ◽  
Annexin Iv

The annexins are Ca(2+)-regulated, phospholipid-binding proteins which have been suggested to take part in cellular events such as exocytosis. The subcellular localization of annexins in human neutrophils was determined using monoclonal antibodies against annexins I, II, IV and VI and a polyclonal peptide antiserum against an annexin consensus sequence. Several annexins were translocated to the light membrane fraction enriched in plasma membranes and secretory vesicles. Annexins were associated also with the azurophil and specific granules. Whereas annexins I, IV and VI and one unidentified 35 kDa protein translocated to each of the isolated organelles, annexin II, a 66 kDa annexin IV-like protein, and a 38 kDa annexin I-like protein exhibited organelle-related differences in their association with membranes. The 38 kDa annexin associated only with specific granules and the secretory vesicles/plasma membrane but not with azurophil granules. Annexin II and the 66 kDa annexin IV-like protein associated with each of the neutrophil organelles, but the binding to specific granules and secretory vesicles/plasma membrane showed a Ca(2+)-dependency different from that of azurophil granules. This observation suggests that these proteins may contribute to the secretory process in neutrophils.

scholarly journals Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst

1981 ◽  
Vol 194 (2) ◽  
pp. 599-606 ◽  
Author(s):  
A R Cross ◽  
O T Jones ◽  
A M Harper ◽  
A W Segal
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Plasma Membranes ◽  
Mixing Time ◽  
Human Neutrophils ◽  
Half Time ◽  
Sulphur Compounds ◽  
Midpoint Potential ◽  
Oxidation Reduction ◽  
Membrane Bound

The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.

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