Mechanisms of hydrogen peroxide formation by leukocytes

Takanaka, Koichiro (1975) Mechanisms of hydrogen peroxide formation by leukocytes. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

It is postulated that the increase in H₂O₂ formation following phagocytosis in guinea pig polymorphonuclear leukocytes is due to the activation of a plasma membrane located NAP(P)H oxidase. -- The cyanide-resistant oxidase activity of intact leukocytes was markedly stimulated when the leukocytes were suspended in a hypotonic medium. Hydrogen peroxide was the principal product of the oxidase reaction. Furthermore the oxidase was active enough to account for the respiratory burst that accompanies phagocytosis. Evidence that the oxidase activity was located on the outside surface of the plasma membrane was the finding that added NAD(P)H was rapidly oxidized and the plasma membrane was impermeable to NADH or NADPH. Further evidence was the marked inhibition of the oxidase by p-CMB which also did not penetrate the plasma membrane. The oxidase was also inhibited on disruption of the plasma membrane. In addition, the enhanced oxidase activity under hypotonic conditions decreased to normal values when the medium was made isotonic and suggested that a reversible conformational change in the plasma membrane was responsible for the activation of oxidase activities. -- Treatment of the leukocytes with digitonin at concentrations low enough so that the leukocytes were not disrupted caused a cyanide-resistant burst of oxygen consumption. Added cytochrome c was reduced and this reduction was blocked by superoxide-dismutase, indicating the formation of superoxide radicals by the leukocytes during the burst of oxygen consumption. -- The cyanide-resistant oxygen consumption of resting PMNs was also found to be stimulated by 2,4-dichlorophenol with H₂O₂ being the sole product formed. NADH and NADPH added to the leukocytes greatly enhanced the oxygen consumption and were oxidized in the process without penetrating the leukocytes. Mn²⁺ stimulated this oxidase activity. Purified peroxidases also had NAD(P)H oxidase activity with similar properties. The NADPH oxidase activity of polymorphonuclear leukocyte granules has not previously been attributed to myeloperoxidase because of its relative insensitivity to cyanide and its activation by aminotriazole. However, the NAD(P)H oxidase activity of myeloperoxidase or horseradish peroxidase was little affected by 2.0 mM cyanide although the peroxidase activity was nearly completely inhibited by 0.1 mM cyanide. Furthermore, the NAD(P)H oxidase activity of myeloperoxidase was considerably enhanced by aminotriazole although the peroxidase activity was inhibited. However, myeloperoxidase is not responsible for the oxidase activity of intact leukocytes as the peroxidase activity of intact leukocytes was very low and density gradient fractionation of the leukocytes showed that the NAD(P)H oxidase activity of the cytosol fraction had very low peroxidase activity.

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