Use of an Affinity Label to Probe the Function of the NADPH Binding Component of the Respiratory Burst Oxidase of Human Neutrophils

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.

Download Full-text

The respiratory burst oxidase of human neutrophils

Free Radical Biology and Medicine ◽

10.1016/0891-5849(90)90668-9 ◽

1990 ◽

Vol 9 ◽

pp. 139

Author(s):

Bernard M. Babior

Keyword(s):

Respiratory Burst ◽

Human Neutrophils ◽

Respiratory Burst Oxidase

Download Full-text

Evidence for participation of vicinal dithiols in the activation sequence of the respiratory burst of human neutrophils

Blood ◽

10.1182/blood.v85.9.2559.bloodjournal8592559 ◽

1995 ◽

Vol 85 (9) ◽

pp. 2559-2569 ◽

Cited By ~ 27

Author(s):

H Kutsumi ◽

K Kawai ◽

RB Jr Johnston ◽

K Rokutan

Keyword(s):

Tyrosine Phosphorylation ◽

Respiratory Burst ◽

Tyrosine Phosphatase ◽

Human Neutrophils ◽

Free System ◽

Kinase C ◽

Ring Complex ◽

A Cell ◽

Respiratory Burst Oxidase ◽

Vicinal Dithiols

Phenylarsine oxide (PAO) specifically forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP). Pretreatment of human neutrophils with micromolar concentrations of PAO inhibited release of superoxide anion (O2-) stimulated by N- formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA); the inhibition was reversed with DMP, but not with 2-mercaptoethanol. PAO did not affect O2-release in previously stimulated cells. PAO did not affect the FMLP-induced Ca2+ response, suggesting that PAO affects a postreceptor event that does not modulate the Ca2+ transient. Treatment of isolated membrane or cytosolic fractions with PAO did not change the rates of arachidonate-stimulated O2-production in a cell-free system. Pretreatment of unstimulated neutrophils with PAO inactivated cytosolic protein kinase C (PKC); the inactivation was reversed with DMP. However, PAO did not affect PMA-induced translocation of beta-PKC protein or reduce the PKC activity translocated to the membrane. PAO had no effect on tyrosine kinase activity but inactivated phosphotyrosine phosphatase; stimulus-induced tyrosine phosphorylation of several proteins was markedly enhanced. These results suggest that vicinal dithiols play an essential role in activation of the respiratory burst oxidase. Possible sites for the activity of these essential vicinal dithiols include PKC and the regulatory balance of tyrosine phosphatase activity and tyrosine phosphorylation.

Download Full-text