Competitive and noncompetitive inhibition of the DNA-dependent protein kinase.
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The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase that is involved in mammalian DNA double-strand break repair. The catalytic subunit of DNA-PK (DNA-PKcs) shares sequence homology in its kinase domain with phosphatidylinositol (PI) 3-kinase. Here, we provide a detailed kinetic analysis of DNA-PK inhibition by the PI 3-kinase inhibitor wortmannin and demonstrate this inhibition to be of a noncompetitive nature, with a Ki of 120 nM. Another inhibitor of PI 3-kinase. LY294002, its parent compound, quercetin, and other derivatives have also been studied. These chemicals are competitive inhibitors of DNA-PK, with LY294002 having a Ki of 6.0 microM. Using an antibody to wortmannin, we found that this compound binds covalently to the kinase domain of DNA-PKcs both in vitro and in vivo. Binding of wortmannin to the active site of DNA-PKcs is inhibited by ATP but not by a peptide substrate. Furthermore, wortmannin is able to bind to DNA-PKcs independently of Ku, and it is not stimulated by the presence of DNA. This suggests that the ATP binding site of DNA-PKcs is open constitutively and that DNA activation of the kinase is mediated via another mechanism. (+info)
Part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal lumen.
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Rutin and quercetin absorption and metabolism were investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min). In contrast to rutin, a high proportion of quercetin (two-thirds) disappeared during perfusion, reflecting extensive transfer into the intestinal wall. Net quercetin absorption was not complete (2.1 nmol/min), inasmuch as 52% were reexcreted in the lumen as conjugated derivatives (7.7 nmol/min). Enterohepatic recycling contribution of flavonoids was excluded by catheterization of the biliary duct before perfusion. After a 30-min perfusion period, 0.71 microM of quercetin equivalents were detected in plasma, reflecting a significant absorption from the small intestine. The differential hydrolysis of effluent samples by glucuronidase and/or sulfatase indicates that the conjugated forms released in the lumen were 1) glucuronidated derivatives of quercetin and of its methoxylated forms (64%) and 2) sulfated form of quercetin (36%). In vitro quercetin glucuronides synthetized using jejunal and ileal microsomal fractions were similar to those recovered in the effluent of perfusion. These data suggest that glucuronidation and sulfatation take place in intestinal cells, whereas no glucurono-sulfoconjugates could be detected in the effluent. The present work shows that a rapid quercetin absorption in the small intestine is very effective together with its active conjugation in intestinal cells. (+info)
Theoretical elucidation of activity differences of five phenolic antioxidants.
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AIM: To verify the effectiveness of structure-activity relationship (SAR) and theoretical calculation methods for antioxidants. METHODS: Preliminary elucidation on the differences of activities of 5 antioxidants was performed by SAR. Then semiempirical quantum chemistry method AM1 was employed to calculate the delta HOF value, the difference between the heat of formation of antioxidant and its free radical, which was used as a theoretical parameter to elucidate the differences of activities of the antioxidants thoroughly. RESULTS: delta HOF values of antioxidants were obtained as follows: ferulic acid, 150.58 kJ.mol-1; anion of ferulic acid, 122.64 kJ.mol-1; modified ferulic acid, 137.70 kJ.mol-1; anion of modified ferulic acid, 118.99 kJ.mol-1; salvianic acid, 134.17 kJ.mol-1; rutin, 137.83 kJ.mol-1, L-EGCG, 124.39 kJ.mol-1; paeonol, 176.79 kJ.mol-1. The differences of the antioxidant activities were elucidated, and how to further enhance the antioxidant activity was investigated as well. CONCLUSION: The SAR and calculation methods are rather effective to elucidate the differences of antioxidant activities, and present some new clues for structural modification of antioxidants to increase their activities. (+info)
Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein.
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Flavonol 2,4-dioxygenase, which catalyzes the cleavage of quercetin to carbon monoxide and 2-protocatechuoyl-phloroglucinol carboxylic acid, was purified from culture filtrate of Aspergillus niger DSM 821 grown on rutin. It is a glycoprotein (46-54% carbohydrate) with N-linked oligo-mannose type glycan chains. The enzyme was resolved in SDS polyacrylamide gels in a diffuse protein band that corresponded to a molecular mass of 130-170 kDa. When purified flavonol 2,4-dioxygenase was heated, it dissociated into three peptides with apparent molecular masses of 63-67 kDa (L), 53-57 kDa (M), and 31-35 kDa (S), which occurred in a molar ratio of 1:1:1, suggesting a LMS structure. Crosslinking led to a 90-97 kDa species, concomitant with the decrease of staining intensity of the 63-67 kDa (L) and the 31-35 kDa (S) peptides. Analysis by matrix-assisted laser desorption/ionization-time of flight-MS showed peaks at m/z approximately 69 600, m/z approximately 51 700, and m/z approximately 26 500 which are presumed to represent the three peptides of flavonol 2,4-dioxygenase, and a broad peak at m/z approximately 96 300, which might correspond to the LS heterodimer as formed in the crosslinking reaction. Based on the estimated molecular mass of 148 kDa, 1 mol of enzyme contained 1.0-1.6 mol of copper. Ethylxanthate, which specifically reduces CuII to CuI ethylxanthate, is a potent inhibitor of flavonol 2,4-dioxygenase. Metal chelating agents (such as diethyldithiocarbamate, diphenylthiocarbazone) strongly inhibited the enzymatic activity, but inactivation was not accompanied by loss of copper. The EPR spectrum of flavonol 2,4-dioxygenase (as isolated) showed the characteristic parameters of a nonblue type 2 CuII protein. The Cu2+ is assumed to interact with four nitrogen ligands, and the CuII complex has a (distorted) square planar geometry. (+info)
The small intestine can both absorb and glucuronidate luminal flavonoids.
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We have studied the perfusion of the jejunum and ileum in an isolated rat intestine model with flavonoids and hydroxycinnamates and the influence of glycosylation on the subsequent metabolism. Flavone and flavonol glucosides and their corresponding aglycones are glucuronidated during transfer across the rat jejunum and ileum and this glucuronidation occurs without the need for gut microflora. Furthermore, this suggests the presence of glycosidases as well as UDP-glucuronyl transferase in the jejunum. In contrast, quercetin-3-glucoside and rutin are mainly absorbed unmetabolised. The results suggest that the more highly reducing phenolics are absorbed predominantly as glucuronides (96.5%+/-4.6) of the amount absorbed, whereas monophenolic hydroxycinnamates and monophenolic B-ring flavonoids are less predisposed to glucuronidation and higher levels of aglycone (88.1%+/-10.1) are detected on absorption through both the jejunum and ileum. (+info)
A beta-rutinosidase from Penicillium rugulosum IFO 7242 that is a peculiar flavonoid glycosidase.
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A beta-rutinosidase, which was specific for releasing the disaccharide rutinose from the flavonoid glycoside rutin, was purified from Penicillium rugulosum IFO 7242. This enzyme had the molecular weight of 245,000, a very low optimum pH of 2.2, and the remarkable specificity that the glycosidase did not hydrolyze any other substrates like 4-nitrophenyl beta-glucoside and cellobiose, but only rutin and isoquercitrin. (+info)
Reactive oxygen species (ROS) are known to modulate activities of a host of kinases, phosphatases and transcription factors. Rutin and chlorogenic acid (CGA) are the major polyphenolic antioxidants present in the small molecular fraction of smokeless tobacco leaf extracts, as ascertained by reverse-phase high-pressure liquid chromatography (HPLC) and mass spectrometry. Levels of intracellular ROS in resting versus antigen-immunoglobulin E (IgE)-challenged murine mast cells were measured at 510 nm by fluorescence-activated cell sorting (FACS) using carboxy-dichlorofluorescein (DCFH-DA). Enhanced ROS production was observed in IgE-sensitized mast cells following antigenic challenge. Rutin and CGA reduced ROS levels in antigen-IgE-activated mast cells. Concomitantly, they also profoundly inhibited histamine release by these activated mast cells. In contrast, rutin and CGA augmented the inducible cytokine messages, i.e. interleukin (IL)-10, IL-13, interferon-gamma (IFN-gamma), IL-6 and tumour necrosis factor-alpha (TNF-alpha) in IgE-sensitized mast cells following antigen challenge. This study indicates that tobacco polyphenolic antioxidants that quench intracellular ROS, differentially affect two effector functions of antigen-IgE-activated mast cells. This model system may be employed to determine the molecular target of polyphenols. The potential role of these polyphenolic antioxidants on IgE-mediated allergy in vivo depends on a balance of their differential effects on mast cell activation. (+info)
Chemoprevention studies of the flavonoids quercetin and rutin in normal and azoxymethane-treated mouse colon.
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In this study we investigated the chemopreventive effects of quercetin and rutin when added to standard AIN-76A diet and fed to normal and azoxymethane (AOM)-treated mice. Early changes in colonic mucosa were analyzed, including colonic cell proliferation, apoptotic cell death, cyclin D(1) expression and focal areas of dysplasia (FAD). The findings show that the number of colonic epithelial cells per crypt column increased (P: < 0.01) in each normal mouse group fed the flavonoids; AOM administration increased colonic crypt cell proliferation and resulted in a marked rise of bromodeoxyuridine-labeled cells in the lower proliferative zone of the crypt. Both supplementary dietary quercetin and rutin increased the apoptotic index and caused a redistribution of apoptotic cells along the crypt axis in normal mice fed a standard AIN-76A diet. The number of apoptotic cells/column and apoptotic indices markedly increased (P: < 0.01) in the AOM-treated group compared with untreated animals; apoptotic cells expanded throughout the colonic crypts after flavonoid supplementation and AOM administration. Positive cyclin D(1) expression was detected in mice on diets supplemented either with quercetin (P: < 0.01) or rutin (P: < 0.05). AOM administration resulted in the formation of FAD. Both the number of mice exhibiting FAD and the total numer of FAD observed were significantly reduced (P: < 0.01) in AOM-treated animals fed flavonoids compared with mice maintained on the standard AIN-76A diet. Surprisingly, however, quercetin alone was able to induce FAD in 22% of normal mice fed the standard AIN-76A diet. (+info)