scholarly journals The association of FAD with the cytochrome b–245 of human neutrophils

1982 ◽  
Vol 208 (3) ◽  
pp. 759-763 ◽  
Author(s):  
Andrew R. Cross ◽  
Owen T. G. Jones ◽  
Rudolfo Garcia ◽  
Anthony W. Segal
Keyword(s):  
Plasma Membrane ◽  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Membrane Fraction ◽  
Close Association ◽  
Gradient Centrifugation ◽  
Fluorescence Emission ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Two Peaks

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b–245 at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b–245 the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b–245 and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b–245 of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b–245 and support a scheme for electron transport thus: [Formula: see text]

scholarly journals Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2438-2445 ◽  
Author(s):  
MT Quinn ◽  
JT Curnutte ◽  
CA Parkos ◽  
ML Mullen ◽  
PJ Scott ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Human Neutrophil ◽  
Autosomal Recessive ◽  
Plasma Membranes ◽  
Close Association ◽  
Granulomatous Disease ◽  
Detergent System

Abstract Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

scholarly journals The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils

1988 ◽  
Vol 251 (3) ◽  
pp. 887-891 ◽  
Author(s):  
J A Ellis ◽  
S J Mayer ◽  
O T Jones
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Oxidase Inhibitor ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Nadph Oxidase Inhibitor ◽  
Specific Granules ◽  
Aerobic And Anaerobic

NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.

scholarly journals Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2438-2445 ◽  
Author(s):  
MT Quinn ◽  
JT Curnutte ◽  
CA Parkos ◽  
ML Mullen ◽  
PJ Scott ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Human Neutrophil ◽  
Autosomal Recessive ◽  
Plasma Membranes ◽  
Close Association ◽  
Granulomatous Disease ◽  
Detergent System

Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

CYTOCHROME b IS PRESENT IN NEUTROPHILS FROM PATIENTS WITH CHRONIC GRANULOMATOUS DISEASE

The Lancet ◽  
1979 ◽  
Vol 313 (8123) ◽  
pp. 949-951 ◽  
Author(s):  
Niels Borregaard ◽  
KirstenStahr Johansen ◽  
Ebbe Taudorff ◽  
JohanH. Wandall
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Granulomatous Disease

scholarly journals Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

1990 ◽  
Vol 1025 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Joseph W. Francis ◽  
James E. Smolen ◽  
Kenneth J. Balazovich ◽  
Rebecca R. Sandborg ◽  
Laurence A. Boxer
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Human Neutrophils ◽  
Plasma Membrane Fraction ◽  
Calcium Dependent

The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

scholarly journals Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 231-233 ◽  
Author(s):  
PD Lew ◽  
C Wollheim ◽  
RA Seger ◽  
T Pozzan
Keyword(s):  
Chronic Granulomatous Disease ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Granulomatous Disease ◽  
Chemotactic Peptide ◽  
Intact Cells ◽  
Calcium Indicator ◽  
Free Calcium Concentration

Abstract Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

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 Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1416-1420 ◽  
Author(s):  
CA Parkos ◽  
MC Dinauer ◽  
AJ Jesaitis ◽  
SH Orkin ◽  
JT Curnutte
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Autosomal Recessive ◽  
Polyclonal Antibodies ◽  
Granulomatous Disease ◽  
Absorbance Spectrum ◽  
Phagocytic Cells ◽  
Inherited Disorders ◽  
Western Blots ◽  
Respiratory Burst Oxidase

Abstract Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytic cells fail to generate antimicrobial oxidants. The various forms of CGD can be classified in terms of the mode of inheritance (either X-linked or autosomal recessive), and whether the neutrophils display the absorbance spectrum of a unique b-type cytochrome important for the function of the respiratory burst oxidase. The finding that purified neutrophil cytochrome b is a heterodimer consisting of a 91kD glycosylated and a 22kD nonglycosylated polypeptide has raised the question of which subunits are absent (or defective) in the various types of CGD. To address this question we have studied the expression of the cytochrome b subunits in three genetically distinct forms of CGD: X-linked/cytochrome b-negative (X-), autosomal recessive/cytochrome b-negative (A-), and autosomal recessive/cytochrome b-positive (A+). Using polyclonal antibodies to each of the two subunits, we prepared Western blots of lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.

A variant form of X-linked chronic granulomatous disease with normal nitroblue tetrazolium slide test and cytochrome b

1983 ◽  
Vol 13 (3) ◽  
pp. 243-247 ◽  
Author(s):  
NIELS BORREGAARD ◽  
ANDREW R. CROSS ◽  
TROELS HERLIN ◽  
OWEN T. G. JONES ◽  
ANTHONY W. SEGAL ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Nitroblue Tetrazolium ◽  
Variant Form ◽  
Granulomatous Disease ◽  
Slide Test
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