Journal of Biochemistry Advance Access originally published online on October 27, 2007
Journal of Biochemistry 2008 143(1):107-115; doi:10.1093/jb/mvm196
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© 2007 The Japanese Biochemical Society.
A Prodigiosin Analogue Inactivates NADPH Oxidase in Macrophage Cells by Inhibiting Assembly of p47phox and Rac
1NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE); 2Division of Biochemistry, Faculty of Fisheries, Nagasaki University; 3Suntory Institute for Bioorganic Research; 4New Industry Creation Hatchery Center, Tohoku University; 5Institute of Multidisciplinary Research for Advanced Materials, Tohoku University; and 6Joint Research Center, Nagasaki University, Japan
*To whom correspondence should be addressed. Tel: +81-438-20-5764, Fax: +81-438-52-2314, E-mail: nakashima-takuji{at}nite.go.jp
Received August 6, 2007; Accepted October 9, 2007
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Abstract |
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Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O2–) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED50 value was estimated to be 
0.3 µM. PG-L-1 had no direct scavenging effect on O2– generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O2– production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67phox, p47phox, p40phox, Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47phox and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47phox and Rac to the membrane components of NADPH oxidase.
Key Words: NADPH oxidase inhibitor, prodigiosin, p47phox, Rac protein, superoxide
Abbreviations: PMA, phorbol 12-myristate 13-acetate; NADPH, nicotinamide adenine dinucleotide phosphate; phox, phagocytic oxidase; , phosphate; PKC, protein kinase C; ROS, reactive oxygen species; Nox, NADPH oxidase; phox, phagocytic oxidase; CGD, chronic granulomatous disease; Q-TOF, quadrupole time-of-flight; FT-ICR-MS, Fourier transform ion cyclotron resonance mass spectrometer; TMS, Tetramethylsilane; KRP, Krebs-Ringer-Phosphate; ESR, Electron spin resonance; ESI, electrospray ionization
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