Cloning and characterization of a cDNA encoding a novel extracellular peroxidase from Trametes versicolor.
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The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression. (+info)
Separation and properties of two acetylacetoin reductases from Bacillus cereus YUF-4.
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The separation and purification of two kinds of acetylacetoin reductases (AACRs) from Bacillus cereus YUF-4 were examined. NADPH-linked AACR (AACR I) and NADH-linked AACR (AACR II) were separated from each other by ammonium sulfate fractionation, DEAE-cellulose chromatography, and hydroxyapatite chromatography. The former was purified 3.4-fold with a yield of 10.0%, and the latter was purified 29-fold with a yield of 15.6%. The two enzymes differ from each other in some enzymic properties such as substrate specificity. (+info)
Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex.
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We describe a novel and general, mechanism-based, method for purification of xyloglucan endotransglycosylases (XETs) from crude plant extracts. Putative isoforms, obtained by step-wise precipitation with (NH4)2SO4, were incubated with tamarind xyloglucan (approximately 1 MDa) to form stable xyloglucan-XET complexes with apparent molecular masses >500 kDa on gel-permeation chromatography (GPC). Subsequent addition of xyloglucan-derived oligosaccharides (a mixture of XET acceptor substrates) caused a shift in the GPC elution volume of the activity back to that expected of a approximately 32 kDa protein, presumably by completing the transglycosylation reaction and so freeing the enzyme from the xyloglucan (donor substrate). This simple two-step method enabled the isolation of each XET activity attempted [various (NH4)2SO4 cuts from extracts of cauliflower florets and mung bean seedlings], in pure form as judged by SDS/PAGE. (+info)
Differences in phosphofructokinase regulation in normal and tumor rat thyroid cells.
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The kinetic and molecular properties of a phosphofructokinase derived from a transplantable rat thyroid tumor lacking regulatory control on the glycolytic pathway were studied. The properties of the near-purified enzyme (specific activity 140 units/mg) were compared with those of phosphofructokinase from normal rat thyroid (specific activity 134 units/mg). The electrophoretic mobilities and gel elution behavior of these two enzymes were almost similar. The thyroid tumor phosphofructokinase showed, however, a greater degree of size and/or shape heterogeneity in the presence of ATP than the normal thyroid enzyme, as determined by gel filtration and sucrose density gradient centrifugation. Kinetic studies below pH 7.4 showed a sigmoid response curve for both enzymes when the velocity was determined at 1 mM ATP with varying levels of fructose-6-P. The interaction coefficient, however, was 4.2 and 2.6 for normal and tumor thyroid phosphofructokinase, respectively. Ammonium sulfate decreased the cooperative interactions with the substrate fructose-6-P in both enzymes. The thyroid tumor enzyme, however, was less sensitive to the inhibition by ATP and by citrate. The reversal of citrate inhibition by cyclic 3':5'-adenosine monophosphate was also less effective with the thyroid tumor phosphofructokinase, while the protective effect of fructose-6-P was stronger. The difference in citrate inhibition between tumor and normal thyroid enzyme was not strongly affected by varying the MgCl2 concentration up to 10 mM. It is concluded that the complex allosteric regulation typical of the normal thyroid phosphofructokinase is still present in the enzyme isolated from the thyroid tumor tissue. The latter, however, is more loosely controlled by its physiological effectors, such as ATP, citrate, and cyclic AMP. (+info)
Magnesium-dependent folding of self-splicing RNA: exploring the link between cooperativity, thermodynamics, and kinetics.
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Folding of the Tetrahymena self-splicing RNA into its active conformation involves a set of discrete intermediate states. The Mg2+-dependent equilibrium transition from the intermediates to the native structure is more cooperative than the formation of the intermediates from the unfolded states. We show that the degree of cooperativity is linked to the free energy of each transition and that the rate of the slow transition from the intermediates to the native state decreases exponentially with increasing Mg2+ concentration. Monovalent salts, which stabilize the folded RNA nonspecifically, induce states that fold in less than 30 s after Mg2+ is added to the RNA. A simple model is proposed that predicts the folding kinetics from the Mg2+-dependent change in the relative stabilities of the intermediate and native states. (+info)
Selective peroxisome degradation in Yarrowia lipolytica after a shift of cells from acetate/oleate/ethylamine into glucose/ammonium sulfate-containing media.
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We have shown that peroxisomes of the yeast Yarrowia lipolytica are subject to specific degradation after exposure of acetate/oleate-grown cells to glucose excess conditions. Electron microscopic analysis has revealed that the peroxisomes were degraded by uptake in the vacuole. Our results suggest that peroxisomes are taken up by macroautophagic processes, because sequestration of individual peroxisomes, which occurs typically at the beginning of microautophagy, was never observed. The observation that a peroxisomal amine oxidase activity is specifically induced by ethylamine was used for the development of a plate assay screening procedure to isolate peroxisome degradation-defective mutants. (+info)
Complementary effects of bifidogenic growth stimulators and ammonium sulfate in natural rubber serum powder on Bifidobacterium bifidum.
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Natural rubber serum powder, rich in crude protein and carbohydrates, had a strong growth-stimulating activity for Bifidobacterium bifidum JCM 1254, which was unable to grow in a fully synthetic medium, B12 assay medium. Natural rubber serum powder was fractionated by ultrafiltration (molecular weight cutoff 1000). The active ultrafiltrate was further concentrated and desalted with an adsorptive microconcentrator, which adsorbs virtually all amino acids and peptides. Through this purification step, it was found that the adsorbed fraction obtained did not stimulate growth independently but acted complementarily with a small amount of ammonium sulfate. The adsorbed fraction was subsequently analyzed on reversed-phase high pressure liquid chromatography, and the activities of the eluates were measured on B12 assay medium with ammonium sulfate. Consequently, it was proved that several peptidic ingredients in the adsorbed fraction increased the growth of B. bifidum. (+info)
Stimulation of peroxidase activity by decamerization related to ionic strength: AhpC protein from Amphibacillus xylanus.
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AhpC protein, purified from Amphibacillus xylanus with a molecular mass of 20.8 kDa, protects cells against oxidation damage. The enzyme catalyses the reduction of hydroperoxides in cooperation with the 55 kDa flavoprotein, A. xylanus NADH oxidase (NADH oxidase-AhpC system). A. xylanus AhpC has two disulfide linkages between monomers and can act in the homodimer form. Gel-filtration column chromatography and dynamic light scattering (DLS) suggest that A. xylanus AhpC also forms a large oligomeric assembly (10-12 mers). A. xylanus AhpC was crystallized and X-ray diffraction data were collected to 3.0 A. The self-rotation function revealed fivefold and twofold axes located perpendicularly to each other, suggesting that the molecular assembly of A. xylanus AhpC is composed of ten monomers. The oligomerization of A. xylanus AhpC is affected by ionic strength in the DLS measurements. The H(2)O(2) reductase activity of the A. xylanus NADH oxidase-AhpC system is also affected by ionic strength, and it was found that the decamerization of AhpC might be required for the activation of the NADH oxidase-AhpC system. (+info)