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Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

Levine AP, Duchen MR, de Villiers S, Rich PR, Segal AW - PLoS ONE (2015)

Bottom Line: Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced.In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly.Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, University College London, London, United Kingdom.

ABSTRACT
The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

No MeSH data available.


Related in: MedlinePlus

Effects of pH on enzymatic activity.The effect of variations in pH on peroxidatic and chlorinating activities of MPO and on the protease activities of cathepsin G and of elastase are shown. Results shown are the mean + SD of at least three separate assays and are expressed as a percentage of the maximal observed activity.
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pone.0125906.g006: Effects of pH on enzymatic activity.The effect of variations in pH on peroxidatic and chlorinating activities of MPO and on the protease activities of cathepsin G and of elastase are shown. Results shown are the mean + SD of at least three separate assays and are expressed as a percentage of the maximal observed activity.

Mentions: The influence of pH on the peroxidatic and chlorinating activities of MPO and on the protease activities of cathepsin G and elastase are shown in Fig 6. Peroxidase and chlorinating activities of MPO were maximal at acid pH, with kcat for TMB oxidation and MCD chlorination s-1 at pH 5.0 being 36 and 19, respectively. Both activities fell off substantially with elevated pH until virtually nothing of either could be detected at pH 9 or 10. By contrast, activity of both cathepsin G and elastase was low at pH 5, and that for cathepsin G peaked at between 7 and 9 whereas elastase was most active between 8 and 10.


Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

Levine AP, Duchen MR, de Villiers S, Rich PR, Segal AW - PLoS ONE (2015)

Effects of pH on enzymatic activity.The effect of variations in pH on peroxidatic and chlorinating activities of MPO and on the protease activities of cathepsin G and of elastase are shown. Results shown are the mean + SD of at least three separate assays and are expressed as a percentage of the maximal observed activity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401748&req=5

pone.0125906.g006: Effects of pH on enzymatic activity.The effect of variations in pH on peroxidatic and chlorinating activities of MPO and on the protease activities of cathepsin G and of elastase are shown. Results shown are the mean + SD of at least three separate assays and are expressed as a percentage of the maximal observed activity.
Mentions: The influence of pH on the peroxidatic and chlorinating activities of MPO and on the protease activities of cathepsin G and elastase are shown in Fig 6. Peroxidase and chlorinating activities of MPO were maximal at acid pH, with kcat for TMB oxidation and MCD chlorination s-1 at pH 5.0 being 36 and 19, respectively. Both activities fell off substantially with elevated pH until virtually nothing of either could be detected at pH 9 or 10. By contrast, activity of both cathepsin G and elastase was low at pH 5, and that for cathepsin G peaked at between 7 and 9 whereas elastase was most active between 8 and 10.

Bottom Line: Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced.In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly.Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, University College London, London, United Kingdom.

ABSTRACT
The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

No MeSH data available.


Related in: MedlinePlus