Respiratory burst oxidase activation can be dissociated from phosphatidylinositol bisphosphate degradation in a cell-free system from human neutrophils.
Activation of the respiratory burst oxidase in cell-free preparations from 32P-labeled neutrophils was compared with changes in levels of radioactively labeled phosphoinositides in the same preparations. With membrane particles, treatment with sodium dodecyl sulfate (SDS) in the presence of cytosol led to activation of the oxidase without an alteration in levels of labeled phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol 4-phosphate (PIP). Conversely, solubilization of the membrane particles with deoxycholate resulted in loss of nearly 98% of the radioactive PIP2 without activation of the oxidase. In this solubilized preparation, the oxidase could subsequently be fully activated by SDS in the presence of cytosol, even though the labeled PIP2 was almost totally depleted. Two PIP2-derived second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, as well as the protein kinase C activator phorbol myristate acetate (PMA), failed to activate the oxidase. These results suggest that in a cell-free preparation from human neutrophils, detergent-mediated activation of the respiratory burst oxidase is independent of changes in the levels of phosphoinositides or phosphoinositide-derived second messengers.
Traynor
A E
AE
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Scott
P J
PJ
Harris
A L
AL
Badwey
J A
JA
Sklar
L A
LA
Babior
B M
BM
Curnutte
J T
JT
D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000032:analysis; D003902:Detergents; D004789:Enzyme Activation; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013347:Subcellular Fractions; D013481:Superoxides / Q000096:biosynthesis
D003902:Detergents; D008563:Membrane Lipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013481:Superoxides; D012967:Sodium Dodecyl Sulfate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme