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Cloning of rat p47phox and comparison with human p47phox
Michiharu Tanabe1, Olof Rådmark, Takashi Watanabe
1Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77, Stockholm, Sweden. veda@grape.med.tottori-u.ac.jp
Abstract:
A cDNA encoding rat p47phox was cloned from rat spleen cDNA library, utilizing rapid amplification of cDNA ends. The open reading frame corresponded to 389 amino acids: It contained the phagocyte oxidase homology domain, two Src homology 3 domains and a proline rich region, all of which are conserved in mammalian p47phox sequences. Rat p47phox displayed the highest degree of identity to mouse p47phox (94%). We expressed and purified rat p47phox as a glutathione S-transferase fusion protein, and found that the rat protein could replace human p47phox in a cell-free activation system for human NADPH oxidase, giving about half activity. Although rat 12-lipoxygenase interacted with human p47phox in a yeast two-hybrid system, this was not the case for rat p47phox.
