scholarly journals Superoxide-forming enzyme from human neutrophils: evidence for a flavin requirement

Blood ◽  
1977 ◽  
Vol 50 (3) ◽  
pp. 517-524
Author(s):  
BM Babior ◽  
RS Kipnes
Keyword(s):  
Chronic Granulomatous Disease ◽  
Flavin Adenine Dinucleotide ◽  
Flavin Mononucleotide ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Assay Mixture ◽  
Triton X

The superoxide-forming activity of 27,000-g particles prepared from homogenates of zymosan-treated human neutrophils is lost if the assay is conducted in the presence of 0.045% Triton X-100. This loss in activity in the presence of detergent is prevented by 40 micron flavin adenine dinucleotide (FAD), but not by flavin mononucleotide, riboflavin, adenosine 5′-diphosphate, or adenosine 5′-monophosphate. With resting particles or particles from zymosan-treated chronic granulomatous disease neutrophils, no superoxide-forming activity is detectable even in the presence of FAD; this is true whether or not detergent is present in the assay. Particles extracted with detergent prior to assay are fully active if assayed in the presence of FAD, but show little activity if FAD is omitted from the assay mixture. These results suggest that the superoxide-forming enzyme from human neutrophils is a FAD-requiring enzyme.

scholarly journals Superoxide-forming enzyme from human neutrophils: evidence for a flavin requirement

Blood ◽  
1977 ◽  
Vol 50 (3) ◽  
pp. 517-524 ◽  
Author(s):  
BM Babior ◽  
RS Kipnes
Keyword(s):  
Chronic Granulomatous Disease ◽  
Flavin Adenine Dinucleotide ◽  
Flavin Mononucleotide ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Assay Mixture ◽  
Triton X

Abstract The superoxide-forming activity of 27,000-g particles prepared from homogenates of zymosan-treated human neutrophils is lost if the assay is conducted in the presence of 0.045% Triton X-100. This loss in activity in the presence of detergent is prevented by 40 micron flavin adenine dinucleotide (FAD), but not by flavin mononucleotide, riboflavin, adenosine 5′-diphosphate, or adenosine 5′-monophosphate. With resting particles or particles from zymosan-treated chronic granulomatous disease neutrophils, no superoxide-forming activity is detectable even in the presence of FAD; this is true whether or not detergent is present in the assay. Particles extracted with detergent prior to assay are fully active if assayed in the presence of FAD, but show little activity if FAD is omitted from the assay mixture. These results suggest that the superoxide-forming enzyme from human neutrophils is a FAD-requiring enzyme.

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 NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558.

1994 ◽  
Vol 179 (1) ◽  
pp. 291-297 ◽  
Author(s):  
S Tsunawaki ◽  
H Mizunari ◽  
H Namiki ◽  
T Kuratsuji
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Beta Subunit ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Oxidase System ◽  
Cytochrome B558 ◽  
Respiratory Burst Oxidase ◽  
Stimulation Of ◽  
Binding Component

The NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the beta-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32P]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2(-)-generating activity and caused considerable translocation of the cytosolic components p47phox and p67phox. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and approximately 32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the beta-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91phox-deficient X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system.

scholarly journals A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3309-3315 ◽  
Author(s):  
Juan D. Matute ◽  
Andres A. Arias ◽  
Nicola A. M. Wright ◽  
Iwona Wrobel ◽  
Christopher C. M. Waterhouse ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Oxidase Activity ◽  
Autosomal Recessive ◽  
Premature Stop Codon ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Recessive Mutations ◽  
Px Domain

Abstract Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

scholarly journals Altered expression of neutrophil peripheral benzodiazepine receptor in X-linked chronic granulomatous disease

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 184-188
Author(s):  
F Zavala ◽  
F Veber ◽  
B Descamps-Latscha
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Covalent Binding ◽  
Benzodiazepine Receptor ◽  
Mononuclear Phagocytes ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Peripheral Benzodiazepine Receptor ◽  
Cytochrome B558

This study was aimed at determining whether the peripheral benzodiazepine receptor (PBZDR), which is abundantly expressed on mononuclear phagocytes, is involved in host defense mechanisms depending on phagocyte membrane-associated NADPH-oxidase complex. Analysis by reversible and covalent binding of PBZDR expression on human neutrophils shows that it is modulated during NADPH-oxidase activation with phorbol 12-myristate 13-acetate. Based on a series of 17 patients with chronic granulomatous disease (CGD), results show that PBZDR expression is dramatically impaired in X-linked CGD, an inherited disorder due to a mutation on the gene coding for cytochrome b558 NADPH- oxidase component, whereas it is unaffected in autosomal recessive CGD where cytochrome b558 is normally expressed, suggesting a link between PBZDR and cytochrome b558 expressions. PBZDR can be assigned by covalent binding to an 18-Kd membrane protein. These results suggest that the neutrophil PBZDR, which can accommodate the widely prescribed anxiolytic drug Valium (diazepam), is involved in host defense against pathogens, a function that could be affected by neuroimmune interactions.

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

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 Monoclonal antibody 7D5 raised to cytochrome b558 of human neutrophils: immunocytochemical detection of the antigen in peripheral phagocytes of normal subjects, patients with chronic granulomatous disease, and their carrier mothers

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1404-1408 ◽  
Author(s):  
M Nakamura ◽  
M Murakami ◽  
T Koga ◽  
Y Tanaka ◽  
S Minakami
Keyword(s):  
Chronic Granulomatous Disease ◽  
Human Peripheral Blood ◽  
Normal Subjects ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Cytochrome B558 ◽  
Normal Human ◽  
Male Patients ◽  
Normal Human Peripheral Blood ◽  
Cytochrome P 450

We have established a monoclonal hybridoma clone that produces IgG1 against the cytochrome b558 of human neutrophils. The antibody 7D5, secreted by the hybridoma, bound to solubilized cytochrome b of the neutrophils but not to other proteins such as hemoglobin, myeloperoxidase, and pig cytochrome P-450. Immunocytochemical studies of normal human peripheral blood showed that 7D5 bound to neutrophils and monocytes but not to lymphocytes or erythrocytes. The neutrophils of male patients but not of a female patient with chronic granulomatous disease lacked the antigen of 7D5 as well as the absorption spectrum for cytochrome b558. A mosaic of the antigen-positive and -negative neutrophils was observed in mothers of the male patients. These biochemical and immunocytochemical results indicate that 7D5 is a specific antibody against cytochrome b558 of human phagocytes.

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