Cloning of rat p47phox and comparison with human p47phox.
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.
Tanabe
Michiharu
M
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-171 77, Stockholm, Sweden. veda@grape.med.tottori-u.ac.jp
Rådmark
Olof
O
Watanabe
Takashi
T
Shiose
Akira
A
Sumimoto
Hideki
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1