title: Purification and characterization of a lipid thiobis ester from human neutrophil cytosol that reversibly deactivates the O2- -generating NADPH oxidase.
abstract: Intact neutrophils possess a cellular mechanism that efficiently deactivates the microbicidal O2-generating NADPH oxidase during the respiratory burst (Akard, L. P., English, D., and Gabig, T. G. (1988) Blood 72, 322-327). The present studies directed at identifying the molecular mechanism(s) involved in NADPH oxidase deactivation showed that a heat- and trypsin-insensitive species in the cytosolic fraction from normal unstimulated neutrophils was capable of deactivating the membrane-associated NADPH oxidase isolated from opsonized zymosan- or phorbol 12-myristate 13-acetate-stimulated neutrophils. This cytosolic species also deactivated the cell-free-activated oxidase. Deactivation by this cytosolic species occurred in the absence of NADPH-dependent catalytic turnover and was reversible, since NADPH oxidase activity could be subsequently reactivated in the cell-free system. The sedimentable particulate fraction from unstimulated neutrophils did not demonstrate deactivator activity. Deactivator activity was demonstrated in the neutral lipid fraction of neutrophil cytosol extracted with chloroform:methanol. Following complete purification of cytosolic deactivator activity by thin layer chromatography and reversed phase high performance liquid chromatography, the deactivator species was shown to be a lipid thiobis ester compound by mass spectroscopy. Cellular metabolism of this compound in human neutrophils may reveal a unique mechanism for enzymatic control of the NADPH oxidase system and thereby play an important role in regulation of the inflammatory response.
authors.affiliation: Division of Hematology-Oncology, Indiana University School of Medicine, Indianapolis 46202-5121.
authors.forename: E A
authors.initials: EA
authors.lastname: Eklund
authors.forename: T G
authors.initials: TG
authors.lastname: Gabig
mesh_terms: D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011422:Propionates / Q000097:blood* / Q000302:isolation & purification; D013440:Sulfides / Q000097:blood* / Q000302:isolation & purification; D013481:Superoxides / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
chemical_list: D011422:Propionates; D013440:Sulfides; D013481:Superoxides; C064208:propanoic acid 3,3'-thiobis(didodecyl) ester; D015054:Zymosan; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D013755:Tetradecanoylphorbol Acetate