A fusion protein between rac and p67phox (1 - 210) reconstitutes NADPH oxidase with higher activity and stability than the individual components

K. Miyano, S. Ogasawara, C. H. Han, H. Fukuda, M. Tamura

Research output: Contribution to journalArticlepeer-review

37 Citations (Scopus)


Activation of the phagocyte NADPH oxidase, a superoxide-generating enzyme, involves assembly of cytosolic p47phox, p67phox, and rac with the membrane-associated cytochrome b558. Following cell-free activation, enzymatic activity is highly labile [Tamura, M., Takeshita, M., Cumutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529 - 7538]. In an attempt to stabilize the activity and to investigate the nature of the complex, we have produced fusion proteins between rac and a C-terminal truncated form of p67phox (residues 1 - 210, 67N), which is a minimal active fragment. In a cell-free system, a fusion protein 67N-rac had higher activity and a 3-fold higher affinity than the individual cytosolic proteins, and 67N-Ser3-rac, which has a longer linker, showed a similar activity with the individual proteins. In contrast, rac-67N, a fusion in the opposite orientation, showed considerably lower activity. The enzyme activity reconstituted with 67N-rac showed a 10-fold higher stability and a lower Km for NADPH than the individual components. In the absence of p47, 67N-rac fusion protein at a high concentration showed nearly full activation, which was higher than that with the individual components. These results indicate that covalent binding between p67N and rac in the correct order produces a more stable complex than the individual components, suggesting that interactions among the subunits significantly influence the duration of the oxidase activation. On the basis of these findings, we propose a model for the topology among rac, 67N, and cytochrome b558.

Original languageEnglish
Pages (from-to)14089-14097
Number of pages9
Issue number46
Publication statusPublished - Nov 20 2001

All Science Journal Classification (ASJC) codes

  • Biochemistry


Dive into the research topics of 'A fusion protein between rac and p67<sup>phox</sup> (1 - 210) reconstitutes NADPH oxidase with higher activity and stability than the individual components'. Together they form a unique fingerprint.

Cite this