Purification and characterization of a lipid thiobis ester from human neutrophil cytosol that reversibly deactivates the O2--generating NADPH oxidase

Elizabeth A. Eklund*, Theodore G. Gabig

*Corresponding author for this work

Research output: Contribution to journalArticle

10 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)8426-8430
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number15
StatePublished - Jun 12 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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