Characterization of the UDP-glucuronosyltransferases involved in the glucuronidation of an antithrombotic thioxyloside in rat and humans. (1/84)

To investigate the glucuronidation on the hydroxyl group of carbohydrate-containing drugs, the in vitro formation of glucuronides on the thioxyloside ring of the antithrombotic drug, LF 4.0212, was followed in rat and human liver microsomes and with recombinant UDP-glucuronosyltransferases (UGT). The reaction revealed a marked regioselectivity in rat and humans. Human liver microsomes glucuronidated the compound mainly on the 2-hydroxyl position of the thioxyloside ring, whereas rat was able to form glucuronide on either the 2-, 3-, or 4- hydroxyl group of the molecule, although to a lower extent. LF 4.0212 was a much better substrate of human UGT than the rat enzyme (Vmax/Km 30.0 and 0.06 microl/min/mg, respectively). Phenobarbital, 3-methylcholanthrene, and clofibrate enhanced the glucuronidation of LF 4.0212 on positions 2, 3, and 4 of the thioxyloside ring, thus indicating that several UGT isoforms were involved in this process. The biosynthesis of the 2-O-glucuronide isomer was catalyzed by the human UGT1A9 and 2B4, but not by UGT1A6 and 2B11. By contrast, the rat liver recombinant UGT1A6 and 2B1 failed to form the 2-O-glucuronide isomers. From all the recombinant UGTs tested, none catalyzed the formation of the 3-O-glucuronide isomer. Interestingly, glucuronidation on the 4-position was found in all the metabolic competent V79 cell lines considered, including the nontransfected V79 cells, suggesting the presence of an endogenous UGT in fibroblasts able to actively glucuronidate the drug. This activity, which was nonsensitive to the inhibitory effect of 7,7,7-triphenylheptanoic acid, a potent UGT inhibitor, could reflect the existence of a different enzyme.  (+info)

Interaction of the lacZ beta-galactosidase of Escherichia coli with some beta-D-galactopyranoside competitive inhibitors. (2/84)

1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (lacZ) beta-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg(2+) by Mn(2+) enhances both longitudinal and transverse relaxation of the methyl groups of the beta-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-beta-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn(2+) ion is located 8-9A (0.8-0.9nm) from the centroid of the trimethylammonium group and 9A (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (K(i) (o) and K(i) (+) respectively) for the isosteric ligands, beta-d-galactopyranosylbenzene and the beta-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=K(i) (+)/K(i) (o)) obtained with 2-(beta-d-galactopyranosyl)-naphthalene and the beta-d-galactopyranosylisoquinolinium ion at pH7 with Mg(2+)-enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg(2+)-enzyme can be described by Q=0.1(1+[H(+)]/4.17x10(-10))/1+[H(+)]/10(-8)). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg(2+)-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg(2+) as well as tyrosine-501.  (+info)

Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside. (3/84)

2-Nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside (azidophenylgalactoside) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 75 muM. The initial rate and steady state level of [3H]azidophenylgalactoside accumulation are markedly stimulated by the addition of D-lactate to vesicles containing the lac transport system, and kinetic studies reveal an apparent Km of 75 muM. Membrane vesicles devoid of the lac transport system do not take up significant amounts of azidophenylgalactoside in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, azidophenylgalactoside irreversibly inactivates the lac transport system. Strikingly, photolytic inactivation is not observed in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 77 muM. Since lactose protects against inactivation and azidophenylgalactoside does not inactivate amino acid transport, it is apparent that these effects are specific for the lac transport system. The results are consistent with the proposal that the lac carrier protein is inaccessible to substrate in the absence of energy coupling.  (+info)

Effect of hydrogen peroxide treatment on the nutritional quality of rapeseed flour fed to weanling rats. (4/84)

The effect of treating rapeseed flours with hydrogen peroxide on the glucosinolate content and nutritional value of the protein was examined. Four flours were prepared from Target variety rapeseed (Brassica napus) by dehulling and defatting the seed (sample RF), by heat treating and water washing the dehulled seed prior to defatting (sample WWRF), and by treating a part of samples RF and WWRF with solutions of 7 and 3% hydrogen peroxide, respectively. Chemical analysis showed that the hydrogen peroxide treatment lowered the glucosinolate content of the flour but was not as effective as the water extraction. The hydrogen peroxide treatment also oxidized methionine to its sulfoxide and sulfone and cysteine to cysteic acid. In the first experiment, weanling rats were fed for 3 weeks diets in which casein or each of the flour preparations provided 5, 10, or 20% protein. Rats fed the high glucosinolate-containing flour (sample RF) at the 10 to 20% protein level died, while those fed 5% survived but lost weight. Those fed the hydrogen peroxide-treated flours survived, but weight gains and food consumption were low compared with the values of the caseinor WWRF-fed groups. Rats fed sample RF exhibited enlarged thyroids. Those fed the peroxide-treated samples had high plasma levels of methionine sulfoxide and sulfone. In the second experiment, additions of 0.15 or 0.30% methionine to the 10% protein diets resulted in increased weight gains of the groups fed the peroxide-treated flours. It was concluded that the hydrogen peroxide treatment was effective in reducing the glucosinolate content of the rapeseed flour. However, the production of the oxidized sulfur amino acids, in particular methionine sulfone, reduced considerably the nutritional value of the protein.  (+info)

Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis. (5/84)

BACKGROUND: In global gene expression profiling experiments, variation in the expression of genes of interest can often be hidden by general noise. To determine how biologically significant variation can be distinguished under such conditions we have analyzed the differences in gene expression when Bacillus subtilis is grown either on methionine or on methylthioribose as sulfur source. RESULTS: An unexpected link between arginine metabolism and sulfur metabolism was discovered, enabling us to identify a high-affinity arginine transport system encoded by the yqiXYZ genes. In addition, we tentatively identified a methionine/methionine sulfoxide transport system which is encoded by the operon ytmIJKLMhisP and is presumably used in the degradation of methionine sulfoxide to methane sulfonate for sulfur recycling. Experimental parameters resulting in systematic biases in gene expression were also uncovered. In particular, we found that the late competence operons comE, comF and comG were associated with subtle variations in growth conditions. CONCLUSIONS: Using variance analysis it is possible to distinguish between systematic biases and relevant gene-expression variation in transcriptome experiments. Co-variation of metabolic gene expression pathways was thus uncovered linking nitrogen and sulfur metabolism in B. subtilis.  (+info)

Hydrolysis of aryl beta-D-glucopyranosides and beta-D-xylopyranosides by an induced beta-D-glucosidase from Stachybotrys atra. (6/84)

The induced beta-D-glucosidase from Stachybotrys atra hydrolyzes aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by the same basic two-step mechanism. In the first step the aglycon group is split of with simultaneous formation of an enzyme-glycosyl complex. In the second step this intermediate complex reacts with water yeilding beta-D-glucose or beta-D-xylose. For beta-D-xyloside hydrolysis each of the two steps is partially rate-controlling, whereas for beta-D-glucoside hydrolysis the second step is rate-limiting. The enzyme is inhibited by high concentrations of substrate and the exact rate-concentration equation is a second-order equation. 1-Thio-beta-D-glycopyranosides with an aromatic aglycon inhibit the reaction in both a competitive and non-competitive way. A tentative mechanism is proposed to explain all types of inhibition. In this mechanism substrates and inhibitors with an aromatic aglycon group bind through hydrophobic forces to the 'aglycon subsite' of the intermediate enzyme-glycosyl complex. Binding of the second substrate molecule or of the inhibitor to this complex does not prevent the reaction of the glycosyl moiety with water, it only decreases the rate of the second step.  (+info)

Recovery of the accumulation ability of thiomethyl-beta-galactoside in Escherichia coli after bacteriophage T4 infection. (7/84)

Effects of UV-irridiated and unirradiated T4 phage infection on the beta-galactoside accumulation ability in Eschericia coli have been examined by the use of 14C-labeled thiomethyl-beta-galactoside (TMG). Under conditions where a synchronous adsorption of phage takes place, the cellular ability for TMG accumulation is found to be largely inhibited immediately after phage adsorption, but it recovers with time to a new level, which is dependent on the multiplicity of infection. When cells are infected with UV-irradiated T4 at the same multiplicity as that of unirradiated phage, the cellular accumulation ability is more severely inhibited and there is no recovery from the inhibition. The recovery process in T4-infected cells is mostly sensitive to puromycin. These results suggest that the initial inhibition of the TMG accumulation ability is probably caused by the adsorption of phage coats, and the subsequent restoration occurs through the action of a phage-directed protein(s). In the recovery process, no new transport system appears to be involved. The restored ability of TMG accumulation is resistant to the action of superinfecting UV phage. However, different mechanisms appear to be operating in T4-infected cells for the establishment of resistance to ghosts and for the recovery from the phage coat-induced inhibition.  (+info)

A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO. (8/84)

The genomes of several nonphotosynthetic bacteria, such as Bacillus subtilis, and some Archaea include genes for proteins with sequence homology to the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO). We found that such a RuBisCO-like protein (RLP) from B. subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway. A growth-defective mutant, in which the gene for this RLP had been disrupted, was rescued by the gene for RuBisCOfrom the photosynthetic bacterium Rhodospirillum rubrum. Thus, the photosynthetic RuBisCO from R. rubrum retains the ability to function in the methionine salvage pathway in B. subtilis.  (+info)