Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.99.1 (NADPH-diaphorase)
 3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of truncated forms of gp91phox were expressed in Escherichia coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin. TRX-gp91phox (306-569), which contains the putative FAD and NADPH binding sites, showed weak NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91phox (304-423) and TRX-gp91phox (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91phox (306-569), and showed the same K(m) for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. In the presence of Rac1, the cytosolic regulatory protein p67phox stimulated the NBT reductase activity, but p47phox had no effect. Truncated p67phox containing the activation domain (residues 199-210) [C.-H. Han, J.R. Freeman, T. Lee, S.A. Motalebi, and J.D. Lambeth (1998) J. Biol. Chem. 273, 16663-16668] stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: (i) TRX-gp91phox (306-569) contains binding sites for both pyridine and flavin nucleotides; (ii) this flavoprotein domain shows weak diaphorase activity; and (iii) the flavin-binding domain of gp91phox is the target of regulation by the activation domain of p67phox.
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PMID:Characterization of the flavoprotein domain of gp91phox which has NADPH diaphorase activity. 1127 49

A series of truncated forms of His(6)-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 microM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% alpha-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC(50) values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 microM, respectively, while the K(m) value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 microM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1:1 but neither p67N nor Rac alone showed significant binding.
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PMID:Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex. 1526 May