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

研究成果: ジャーナルへの寄稿記事

12 引用 (Scopus)

抜粋

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.

元の言語英語
ページ(範囲)335-340
ページ数6
ジャーナルEuropean Journal of Biochemistry
187
発行部数2
DOI
出版物ステータス出版済み - 1 1990

All Science Journal Classification (ASJC) codes

  • Biochemistry

フィンガープリント Reconstitution of the partially purified membrane component of the superoxide‐generating NADPH oxidase of pig neutrophils with phospholipid' の研究トピックを掘り下げます。これらはともに一意のフィンガープリントを構成します。

  • これを引用