scholarly journals Phosphorylation of the subunits of cytochrome b-245 upon triggering of the respiratory burst of human neutrophils and macrophages

1988 ◽  
Vol 252 (3) ◽  
pp. 901-904 ◽  
Author(s):  
R C Garcia ◽  
A W Segal
Keyword(s):  
Cytochrome B ◽  
Phorbol Myristate Acetate ◽  
Respiratory Burst ◽  
Time Course ◽  
Autosomal Recessive ◽  
Beta Subunit ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Myristate Acetate ◽  
Oxidase System

Cytochrome b-245, the only clearly identified component of the microbicidal oxidase system of phagocytes, is a heterodimer consisting of a 23 kDa (alpha) and a 76-92 kDa (beta) subunit. This study was conducted to examine whether, in common with a number of proteins, the subunits of the cytochrome were phosphorylated upon activation of the oxidase. Both subunits were phosphorylated after activation of neutrophils or macrophages with phorbol myristate acetate or a phagocytic stimulus, although the time course of this process did not parallel that of the oxidase. Phosphorylation of these proteins was normal in cells from two patients with autosomal recessive chronic granulomatous disease, in whom phosphorylation of a 47 kDa protein is defective.

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 The subcellular particulate NADPH-dependent O2.(-)-generating oxidase from human blood monocytes: comparison to the neutrophil system

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 979-983 ◽  
Author(s):  
AN Chaudhry ◽  
JT Santinga ◽  
TG Gabig
Keyword(s):  
Protein Concentration ◽  
Cell Protein ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Myristate Acetate ◽  
Oxidase System ◽  
Significant Difference ◽  
Normal Human

Abstract Highly purified preparations of normal human monocytes obtained from peripheral blood were shown to contain a subcellular particulate O2.(-)- generating oxidase system. This O2.(-)-generating activity was present in particulate preparations from monocytes that had been previously stimulated with phorbol myristate acetate but was low or absent in control preparations from unstimulated monocytes or stimulated monocytes from a patient with chronic granulomatous disease. In the stimulated preparations from normal monocytes, O2.(-)-generation was linearly proportional to cell protein concentration, insensitive to inhibition by azide, and dependent on NADPH as substrate. These characteristics are similar to the O2.(-)-generating oxidase system from human neutrophils. A significant difference in the apparent Km for NADPH was shown between preparations from stimulated monocytes and neutrophils (monocyte 83 +/- 16 microM, neutrophil 31 +/- 5 microM, mean +/- SE). Additionally, affinity of the stimulated monocyte particulate preparation for NADH was unmeasurably low.

scholarly journals Localization of the 47 kDa phosphoprotein involved in the respiratory-burst NADPH oxidase of phagocytic cells

1989 ◽  
Vol 260 (1) ◽  
pp. 243-248 ◽  
Author(s):  
P G Heyworth ◽  
C F Shrimpton ◽  
A W Segal
Keyword(s):  
Cytochrome B ◽  
Respiratory Burst ◽  
Peptide Mapping ◽  
Granulomatous Disease ◽  
Myristate Acetate ◽  
Phagocytic Cells ◽  
Response Curves ◽  
Respiratory Burst Oxidase ◽  
Cytosolic Factor ◽  
Stimulation Of

A 47 kDa phosphoprotein is involved in the respiratory-burst oxidase of phagocytic cells. After stimulation of neutrophils with phorbol myristate acetate, this phosphoprotein was identified in both the cytosol and membranes. Peptide mapping of the two forms resulted in identical patterns of phosphopeptides. Dose-response curves for accumulation of phosphoprotein in the two sites were very similar, whereas the detection of the phosphoprotein in the cytosol preceded that in the membranes. The membrane-associated 47 kDa phosphoprotein was absent from the neutrophils of patients with X-chromosome-linked chronic granulomatous disease, which lack cytochrome b-245, and intermediate levels were detected in the membranes of their heterozygote carrier mothers. Activation of the neutrophil oxidase system appears to be dependent upon phosphorylation of the cytosolic 47 kDa protein and its association with cytochrome b-245 in the membranes. It is probably the cytosolic factor required for reconstitution of the active oxidase in cell-free systems.

scholarly journals Effect of phorbol myristate acetate on the oxidative metabolism of human polymorphonuclear leukocytes

Blood ◽  
1976 ◽  
Vol 47 (4) ◽  
pp. 545-554 ◽  
Author(s):  
LR DeChatelet ◽  
PS Shirley ◽  
RB Jr Johnston
Keyword(s):  
Phorbol Myristate Acetate ◽  
Oxidative Metabolism ◽  
Polymorphonuclear Leukocytes ◽  
Time Course ◽  
Human Neutrophils ◽  
Hexose Monophosphate ◽  
Myristate Acetate ◽  
Bacterial Killing ◽  
Hexose Monophosphate Shunt ◽  
Human Polymorphonuclear Leukocytes

The addition of 0.1 mug/ml of phorbol myristate acetate (PMA) to a suspension of resting human neutrophils causes a marked stimulation of all aspects of cellular oxidative metabolism normally associated with phagocytosis. PMA induces a greatly increased rate of glucose oxidation via the hexose monophosphate shunt, increased production of superoxide anion and of hydrogen peroxide, increased cellular chemiluminescence, and increased iodination of protein material. The time course of hexose monophosphate shunt activation and of chemiluminescence are similar to those observed following phagocytosis of opsonized zymosan; the levels of activation achieved in all cases approximate those seen following phagocytosis. These phenomena are not simply reflections of altered cellular permeability, since PMA actually inhibits the uptake of radioactive 2-deoxyglucose and of uniformly labeled amino acids. The addition of PMA similarly inhibits the uptake of 14C-labeled bacteria, suggesting a competition between the effect of the chemical and the process of phagocytosis. These results suggest that PMA activates the cell in the same manner as does phagocytosis. This compound should provide a useful tool for elucidating the metabolic events underlying the phenomena of phagocytosis and bacterial killing by polymorphonuclear leukocytes.

Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 811-816 ◽  
Author(s):  
N Okamura ◽  
SE Malawista ◽  
RL Roberts ◽  
H Rosen ◽  
HD Ochs ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Phorbol Myristate Acetate ◽  
Genetic Heterogeneity ◽  
Respiratory Burst ◽  
Granulomatous Disease ◽  
Myristate Acetate ◽  
Straightforward Manner ◽  
Neutral Ph ◽  
The Relationship

Abstract Activation of 32P-loaded neutrophils with phorbol myristate acetate causes the labeling of a family of three 48K proteins that focus near neutral pH. The relationship between these phosphoproteins and the activation of the respiratory burst has been supported by the previous finding that phosphorylation was defective in the two most common types of chronic granulomatous disease (CGD): X-linked cytochrome-negative (X/-) and autosomal cytochrome-positive (A/+). In this report, these studies have now been extended to the rare A/- and X/+ forms of the disease. In all three patients with A/- CGD examined, the two most acidic 48K proteins failed to undergo enhanced phosphorylation in response to phorbol stimulation, a finding similar to that seen in X/- patients. In contrast, neutrophils from two patients with X/+ CGD appeared to phosphorylate the neutral 48K proteins in a normal fashion. It thus appears that the different phosphorylation patterns seen in chronic granulomatous disease are a reflection of the genetic heterogeneity of this disorder. These findings lend further support to the conclusion that the 48K phosphoprotein family is related to the respiratory burst, although not necessarily in a straightforward manner.

scholarly journals Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 811-816
Author(s):  
N Okamura ◽  
SE Malawista ◽  
RL Roberts ◽  
H Rosen ◽  
HD Ochs ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Phorbol Myristate Acetate ◽  
Genetic Heterogeneity ◽  
Respiratory Burst ◽  
Granulomatous Disease ◽  
Myristate Acetate ◽  
Straightforward Manner ◽  
Neutral Ph ◽  
The Relationship

Activation of 32P-loaded neutrophils with phorbol myristate acetate causes the labeling of a family of three 48K proteins that focus near neutral pH. The relationship between these phosphoproteins and the activation of the respiratory burst has been supported by the previous finding that phosphorylation was defective in the two most common types of chronic granulomatous disease (CGD): X-linked cytochrome-negative (X/-) and autosomal cytochrome-positive (A/+). In this report, these studies have now been extended to the rare A/- and X/+ forms of the disease. In all three patients with A/- CGD examined, the two most acidic 48K proteins failed to undergo enhanced phosphorylation in response to phorbol stimulation, a finding similar to that seen in X/- patients. In contrast, neutrophils from two patients with X/+ CGD appeared to phosphorylate the neutral 48K proteins in a normal fashion. It thus appears that the different phosphorylation patterns seen in chronic granulomatous disease are a reflection of the genetic heterogeneity of this disorder. These findings lend further support to the conclusion that the 48K phosphoprotein family is related to the respiratory burst, although not necessarily in a straightforward manner.

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