Reconstitution of the partially purified membrane component of the superoxide‐generating NADPH oxidase of pig neutrophils with phospholipid

Masahiko NOZAKI, Koichiro TAKESHIGE, Hideki Sumimoto, Shigeki MINAKAMI

Research output: Contribution to journalArticle

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Abstract

The NADPH‐dependent superoxide‐generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell‐free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat‐germagglutinin – agarose column chromatography. The chromatography resulted in loss of the O2‐generating activity in the cell‐free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N‐acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre‐mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent‐free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 ± 0.53 nmol/mg protein (mean ± SD, n= 5). The potency of fraction B in the reconstitution of the O2‐generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2‐generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2‐generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p‐chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0–7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 ± 3.5 μmol O2· min−1· mg−1 membrane‐derived protein (mean ± SD, n= 5) which shows that the membrane component was purified about 100‐fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2‐generating NADPH oxidase and its activation in the cell‐free system requires the reconstitution with phospholipids.

Original languageEnglish
Pages (from-to)335-340
Number of pages6
JournalEuropean Journal of Biochemistry
Volume187
Issue number2
DOIs
Publication statusPublished - Jan 1 1990

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NADPH Oxidase
Cell-Free System
Phospholipids
Neutrophils
Swine
Membranes
Chemical activation
Agarose Chromatography
Column chromatography
Flavin-Adenine Dinucleotide
Respiratory Burst
Azides
Chromatography
NADP
Sodium Dodecyl Sulfate
NAD
Cytosol
Sepharose
Dilution
Assays

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Reconstitution of the partially purified membrane component of the superoxide‐generating NADPH oxidase of pig neutrophils with phospholipid. / NOZAKI, Masahiko; TAKESHIGE, Koichiro; Sumimoto, Hideki; MINAKAMI, Shigeki.

In: European Journal of Biochemistry, Vol. 187, No. 2, 01.01.1990, p. 335-340.

Research output: Contribution to journalArticle

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