Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein

Kei Miyano, Hiroyasu Fukuda, Kentaro Ebisu, Minoru Tamura

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47phox, p67phox, and Rac with the membrane-bound flavocytochrome b558. Recently, we have found that p67phox-(1-210) (p67N) fused with p47phox-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t1/2) of the activity reconstituted with wild-type Rac was 12 min at 37 °C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47phox increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t1/2 = 184 min at 37 °C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L ≤ p67N-RacQ61L ≪ p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67phox, Rac, and p47phox is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.

Original languageEnglish
Pages (from-to)184-190
Number of pages7
JournalBiochemistry
Volume42
Issue number1
DOIs
Publication statusPublished - Jan 14 2003
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

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