Showing papers on "Enzyme assay published in 1995"
TL;DR: It is concluded that G6PD activity is dispensable for pentose synthesis, but is essential to protect cells against even mild oxidative stress.
Abstract: Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme encoded in mammals by an X-linked gene. It has important functions in intermediary metabolism because it catalyzes the first step in the pentose phosphate pathway and provides reductive potential in the form of NADPH. In human populations, many mutant G6PD alleles (some present at polymorphic frequencies) cause a partial loss of G6PD activity and a variety of hemolytic anemias, which vary from mild to severe. All these mutants have some residual enzyme activity, and no large deletions in the G6PD gene have ever been found. To test which, if any, function of G6PD is essential, we have disrupted the G6PD gene in male mouse embryonic stem cells by targeted homologous recombination. We have isolated numerous clones, shown to be recombinant by Southern blot analysis, in which G6PD activity is undetectable. We have extensively characterized individual clones and found that they are extremely sensitive to H2O2 and to the sulfydryl group oxidizing agent, diamide. Their markedly impaired cloning efficiency is restored by reducing the oxygen tension. We conclude that G6PD activity is dispensable for pentose synthesis, but is essential to protect cells against even mild oxidative stress.
540 citations
TL;DR: Study of a panel of INH‐resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single‐strand‐conformation polymorphism analysis (PCR‐SSCP) to detect mutations in katG found that in most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted.
Abstract: The toxicity of the powerful anti-tuberculosis drug isoniazid (INH) is believed to be mediated by the haem-containing enzyme catalase-peroxidase, encoded by the katG gene of Mycobacterium tuberculosis. Compelling evidence for this was obtained by studying a panel of INH-resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single-strand-conformation polymorphism analysis (PCR-SSCP) to detect mutations in katG. In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted. The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N-terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat liability. The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C-terminal domain but do not noticeably alter either enzyme activity or heat stability.
339 citations
TL;DR: This approach to the studies of enzyme mechanisms is well suited for those who are geared more to the organic chemistry of enzymecatalyzed reactions and who have insights into the chemical machinery of active sites of enzymes.
Abstract: Publisher Summary An enzyme inactivator, in general, is a compound that produces irreversible inhibition of the enzyme— that is, it irreversibly prevents the enzyme from catalyzing its reaction. Irreversible in this context, however, does not necessarily mean that the enzyme activity never returns only that the enzyme becomes dysfunctional for an extended (but unspecified) period of time. A mechanism-based enzyme inactivator, by the definition used here, is a compound that is transformed by the catalytic machinery of the enzyme into a species that acts as an affinity labeling agent, a transition state analog, or a tight-binding inhibitor (either covalent or noncovalent) prior to release from the enzyme. Mechanism-based enzyme inactivation is a powerful tool for the studies of enzyme mechanisms and mechanisms of enzyme inactivation, by small molecules. Mechanistic hypotheses can be tested by appropriate molecular design, utilizing the isotopically labeled analogs, to permit the elucidation of the structures of metabolites produced and to determine the portions of the mechanism-based inactivators that become covalently attached to the target enzyme. This approach to the studies of enzyme mechanisms is well suited for those who are geared more to the organic chemistry of enzymecatalyzed reactions and who have insights into the chemical machinery of active sites of enzymes. The use of mechanism-based enzyme inactivators is yet another of the very important methods in enzymology.
333 citations
TL;DR: In this article, a triazole plant growth regulator, paclobutrazol, was used to reduce photoinhibition and loss of fresh mass, protein, and membrane integrity in the leaves.
195 citations
TL;DR: A thermophilic bacterium isolated from a hot spring area of Yellowstone National Park was isolated and its enzyme activity was stable to both hydrogen peroxide and alkaline protease which are detergent ingredients.
185 citations
TL;DR: The enzyme is absent in the thylakoids of mature green cotyledons and appears upon the induction of foliar senescence and reaches the highest levels towards the end of the yellowing process.
Abstract: Solubilization of senescent thylakoids from rape cotyledons in the presence of Triton X-100 was employed to establish an in vitro system that allowed the assessment of enzymatic conversion of phaeophorbide a into an uncoloured fluorescent chlorophyll catabolite, Bn-FCC-2. The action of the putative dioxygenase responsible for the cleavage of the porphyrin macrocycle depends on reduced ferredoxin as reductant. Apart from this thylakoidal catalyst, stromal protein is also required for the production of FCC-2 in vitro. The cleavage reaction does not occur with phaeophorbide b as substrate. Saturation kinetics with phaeophorbide a as substrate yielded an apparent Km -value of c. 200 μ. The enzyme contains iron as suggested by inhibitory effects of appropriate chelators. Enzyme activity lost upon treatment with bipyridyl was partly restored in the presence of Fe-ions; other metal ions such as Cu, Zn and Co were ineffective. The enzyme is absent in the thylakoids of mature green cotyledons. It appears upon the induction of foliar senescence and reaches the highest levels towards the end of the yellowing process.
181 citations
TL;DR: In this paper, the addition of municipal solid-waste compost to the soil was studied in laboratory incubations and the results showed that the added compost increased enzyme activity in an additive way.
Abstract: Modifications of soil microbiological activity by the addition of municipal solid-waste compost were studied in laboratory incubations. Three composts were compared, one lumbricompost and two classical composts with different maturation times. Organic C mineralization and nine enzyme activities (dehydrogenase, peroxidase, cellulase, β-glucosidase, β-galactosidase, N-acetyl-β-glucosaminidase, protease, amidase, and urease) were determined in the composts and the amended soil. Initial enzyme activities varied in the soil according to the sampling date (winter or summer) and were greater in the composts than in the soil, except for urease. Generally, the youngest compost exhibited greater activity than the oldest one. In the amended soil, the composts did not increase enzyme activity in an additive way. Dehydrogenase, the only strictly endocellular enzyme, was the only one for which the activity in the amended soil increased significantly in proportion to the addition of compost. During the incubations, C mineralization and dehydrogenase activity were significantly correlated, indicating that dehydrogenase was a reliable indicator of global microbial activity. Peroxidase activity in the soil remained constant, but increased in the composts and amended soil. Addition of the oldest compost had no effect on the activity of the C cycle enzymes, but the youngest compost increased creased soil activity at the higher application rate. Enzymes of the N cycle were stimulated by all compost amendments, but the increase was only transient for amidase and urease. Lumbricomposting had no marked effect on compost enzyme activity, either before or during the incubation.
169 citations
TL;DR: The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this l-asparaginase possesses antitumor properties.
Abstract: An l-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein-1 with an overall recovery of 27.2%. The apparent M
r of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38±0.02. It displayed optimum activity at pH 9.0 and 37°C. The enzyme was very specific for l-asparagine and did not hydrolyze L-glutaminate. The K
m of the l-asparaginase was found to be 1.45×10-4
m towards l-asparagine and was competitively inhibited by 5-diazo-4-oxo-l-norvaline (DONV) with a K
i of 0.03mm. Metal ions such as Mn2+, Zn2+, Hg2+, Fe3+, Ni2+, and Cd2+ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.
161 citations
TL;DR: A rapid, accurate, in vitro assay utilizing radioactive S-adenosylmethionine (SAM) has been developed for the methylation of arsenite and monomethylarsonate by rabbit liver methyltransferases, and there is no evidence at the present time for these enzyme activities being on different protein molecules.
Abstract: A rapid, accurate, in vitro assay utilizing radioactive S-adenosylmethionine (SAM) has been developed for the methylation of arsenite and monomethylarsonate (MMA) by rabbit liver methyltransferases. The assay has been validated by separating, identifying, and measuring the products of the reaction using chloroform extraction, ion exchange chromatography, TLC, or HPLC. The enzymes involved in this pathway, arsenite methyltransferase and MMA methyltransferase, have been purified approximately 2000-fold from rabbit liver. After gel electrophoresis, a single band is obtained with both enzyme activities in it. The pH optima for purified arsenite methyltransferase and monomethylarsonic acid methyltransferase are 8.2 and 8.0, respectively. A thiol, S-adenosylmethionine, and arsenite are required for the partially purified arsenite methyltransferase that catalyzes the synthesis of monomethylarsonate. A different enzyme activity that catalyzes the methylation of monomethylarsonate to dimethylarsinate also requires SAM and a thiol. Even though arsenite methyltransferase and monomethylarsonate methyltransferase have different substrates, pH optima, and saturation concentrations for their substrates, whether the two activities are present on one protein molecule or different protein molecules is still uncertain. Both activities have a molecular mass of 60 kDa as determined by gel exclusion chromatography. There is no evidence at the present time for these enzyme activities being on different protein molecules. Neither arsenate, selenate, selenite, or selenide are methylated by the purified enzyme preparations. Results from the use of crude extracts, often called cytosol, to study the properties of these methyltransferases dealing with arsenic species should be viewed with caution since such crude extracts contain inhibiting and other interfering activities.
160 citations
TL;DR: Chitin deacetylase, the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetyl-D-glucosamine in chitin, has been purified to homogeneity from the culture filtrate of the fungus Colletotrichum lindemuthianum and further characterized.
156 citations
TL;DR: It was found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition, and it was proposed that a major function of the large subunit of adenosine 5[prime]-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme.
Abstract: cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.
TL;DR: The relationship between the intemal rotational mobility of the fluorophore and the enantioselectivity provides the first experimental evidence that enzyme flexibility and enzyme enantiOSElectivity are correlated.
Abstract: A time-resolved fluorescence anisotropy study on the molecular flexibility of active-site labeled anthraniloyl-α-chymotrypsin, dansylsubtilisin Carlsberg, and native subtilisin Carlsberg, suspended in organic solvents, is described. The internal rotational mobility of the fluorophore in the nanosecond time range could be separated from rotation of enzyme aggregates and rapid energy transfer processes. The enzymes suspended in dry organic solvents are less flexible than when dissolved in water. The enzyme flexibility increased with increasing hydration level. The results confirm that the increase in enzyme activity observed upon addition of low amounts of extra water is related to an increase in enzyme flexibility. Differences in enantioselectivity of subtilisin Carlsberg in different organic solvents have been correlated with differences in enzyme flexibility. The relationship between the intemal rotational mobility of the fluorophore and the enantioselectivity provides the first experimental evidence that enzyme flexibility and enzyme enantioselectivity are correlated.
TL;DR: Northern blot analysis of various mouse tissues with the enzyme cDNA as a probe indicated that expression of NeuGc is related to the level of CMP-NeuAc hydroxylase mRNA, and cross-hybridizing bands were detected in the human and fish genomes.
TL;DR: In this article, peroxidase from strawberry fruit was detected, partially purified, and characterized, and the total enzyme extract (both soluble and associated to membranes) was partially purified by means of (NH 4 ) 2 SO 4 precipitation, molecular exclusion chromatography, and cationic exchange chromatography.
Abstract: In this work, peroxidase from strawberry fruit was detected, partially purified, and characterized. The total enzyme extract (both soluble and associated to membranes) was partially purified by means of (NH 4 ) 2 SO 4 precipitation, molecular exclusion chromatography, and cationic exchange chromatography. The purification grade achieved was near 35. Effects of temperature and pH, stability against pH, and thermal stability were analyzed on both crude and partially purified extracts. The maximum enzyme activity was observed at 30 °C and pH 6.0. The enzyme showed low thermal stability and maintained activities equal to or greater than 50% of its maximum value in the 4-11 pH range. Two peroxidase isoenzymes were detected in strawberry fruit ; they were of the basic type (isoelectric points 9.5-10.0) and had molecular masses of 58.1 and 65.5 kDa. Strawberry fruit peroxidase activity decreased remarkably as the fruit ripened and was found primarily in a membrane-bound form. Maximum specific activities were found at the small green and large green ripening stages.
TL;DR: This chapter discusses approaches to measure the hemes spectrally and assay enzyme activity in tissue extracts to characterize the subunit composition of the enzyme to look for alterations in any subunit based on their migration in sodium dodecyl sulfate-polyacrylamide gels.
Abstract: Publisher Summary To explore the biogenesis of mammalian cytochrome-c oxidase and to examine the defects of this enzyme in human disease, approaches are required to measure the hemes spectrally and assay enzyme activity in tissue extracts. This chapter discusses these approaches. It is important to be able to characterize the subunit composition of the enzyme to look for alterations in any subunit based on their migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels, absence of any subunit, and incomplete assembly. The panel of monoclonal antibodies for such studies and their use is described. Several of the monoclonal antibodies will be useful in isolation of the cytochrome-c oxidase complex by immuno-affinity chromatography after covalent binding to Sepharose CL-4B (Pharmacia LKB Biotechnology). This method is established for the purification of the yeast enzyme, using monoclonal antibodies to yeast subunit III, and a similar method is developed for the purification of the human enzyme.
TL;DR: The multifunctional nature cytochrome P-450TYR is confirmed by demonstrating that binding of L-tyrosine or N-hydroxytyrosines mutually excludes binding of the other substrate and the phenomenon of catalytic facilitation (“channeling”) is shown.
TL;DR: 5-Lipoxygenase activity in the human monocytic cell line Mono Mac 6 was upregulated by combined treatment with transforming growth factor beta 1 (TGF-beta) and 1,25-dihydroxyvitamin D3 (VD3) and in undifferentiated cells, 5-LO enzyme activity was undetectable.
Abstract: 5-Lipoxygenase (5-LO; EC 1.13.11.34) activity in the human monocytic cell line Mono Mac 6 was upregulated by combined treatment with transforming growth factor beta 1 (TGF-beta) and 1,25-dihydroxyvitamin D3 (VD3). In undifferentiated cells, 5-LO enzyme activity was undetectable. After the addition of TGF-beta plus VD3, the activity of intact cells was 800 ng per 10(6) cells--500 times more than the assay detection limit. Also 5-LO protein and mRNA expression were induced > 128-fold and 64-fold, respectively, as compared to undifferentiated cells. Both TGF-beta and VD3 were required for these prominent responses. Either agent alone gave small amounts of 5-LO protein and mRNA but very low 5-LO activities. After the addition of TGF-beta and VD3, the induction of 5-LO protein was obvious after 1 day, but the increase in activity was delayed and did not appear until the second day. Pretreatment of cells with TGF-beta or VD3 alone for 2 days led to 5-LO protein expression but very low enzyme activity. Addition of the lacking second inducer was required for full induction of 5-LO protein expression and for upregulation of enzyme activity. Partial purification of 5-LO from Mono Mac 6 cells and recombination with soluble cellular proteins from different sources indicated the presence of cytosolic factors that affect the activity of 5-LO.
TL;DR: The intracellular compartmentation of serine acetyltransferase, a key enzyme in the L-cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts.
Abstract: The intracellular compartmentation of serine acetyltransferase, a key enzyme in the l-cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum)leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in mitochondria (approximately 76% of total cellular activity). Significant activity was also identified in both the cytosol (14 % of total activity) and chloroplasts (10% of total activity). Three enzyme forms were separated by anion-exchange chromatography, and each form was found to be specific for a given intracellular compartment. To obtain cDNA encoding the isoforms, functional complementation experiments were performed using an Arabidopsis thaliana expression library and an Escherichia coli mutant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase isoforms, as confirmed by enzyme activity measurements, genomic hybridizations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino acid sequence shows that it encodes a polypeptide of Mr 34 330 exhibiting 41% amino acid identity with the E. coli serine acetyltransferase. Since none of the general features of transit peptides could be observed in the N-terminal region of this isoform, we assume that it is a cytosolic form.
TL;DR: A 4-hydroxybenzoate decarboxylase from the anaerobe Clostridium hydroxybenzoicum strain JW/Z-1T was purified and partially characterized and the N-terminal amino acid sequence shows some similarity to the uroporphyrinogen decar boxylases from Synechococcus and Saccharomyces.
Abstract: A 4-hydroxybenzoate decarboxylase from the anaerobe Clostridium hydroxybenzoicum strain JW/Z-1T was purified and partially characterized. It had an apparent molecular mass of 350 kDa and consisted of six identical subunits of 57 kDa each. The temperature optimum for the decarboxylation was approximately 50°C, the optimum pH 5.6–6.2. The pI of the enzyme was 5.1. The activation energy for decarboxylation of 4-hydroxybenzoate was 65 kJ · mol−1 (20–37°C). The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate. The apparent Km and kcat values obtained for 4-hydroxybenzoate were 0.40 mM and 3.3×103 min−1, and for 3,4-dihydroxybenzoate 1.2 mM and 1.1×103 min−1, respectively, at pH 6.0 and 25°C. The enzyme activity was not influenced by the addition of biotin or avidin to either the crude cell extracts or the purified enzyme. The p -hydroxyl group of hydroxybenzoate appears to be essential for binding by the enzyme. The N-terminal amino acid sequence shows some similarity to the uroporphyrinogen decarboxylases from Synechococcus and Saccharomyces. The enzyme catalyzed the reverse reactions, that is, the carboxylation of phenol to 4-hydroxybenzoate and of catechol to 3,4-dihydroxybenzoate. The carboxylation did not require ATP.
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31 Dec 1995
TL;DR: Enzyme assays for food scientists, Enzyme Assays for Food scientists, and more.
Abstract: Enzyme assays for food scientists , Enzyme assays for food scientists , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی
TL;DR: This study has demonstrated synthesis and release of gelatinase B by human and rat Kupffer cells in primary culture and characterized the partially purified enzyme, which rapidly degraded denatured collagens (gelatin) as well as native types III, IV, and VCollagens, but had no activity against casein, types I and VI collagen.
TL;DR: Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4- Vinylphenol, respectively, by nonoxidative decarboxylation, and the enzyme responsible for this activity has been purified and characterized.
Abstract: Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.
TL;DR: The pH profile yielded a broadening upon immobilization in addition to a small shift to higher pH (from 5.5 to 6.0).
TL;DR: This is the first biochemical demonstration of the existance of NOS activity in molluscan hemocytes, the cells responsible for defence mechanisms, in Viviparus ater.
TL;DR: Chitin deacetylase, which releases the acetyl groups of glycol chitin was purified from a fungus, Absidia coerulea, and characterized and was demonstrated to be localized near the inner face of the cell wall (periplasmic space) in the mycelia by using immunoelectron microscopy.
Abstract: Chitin deacetylase, which releases the acetyl groups of glycol chitin was purified from a fungus, Absidia coerulea, and characterized. The enzyme was purified 516-fold to homogeneity by means of 65-80% ammonium sulfate precipitation followed by chromatography on Butyl Toyoperal-650M, Gigapite (hydroxyapatite), and DEAE Toyopearl-650M. It had an apparent molecular weight of 75 kDa both on sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration chromatography, indicating that the enzyme exists as a monomer. The amino-terminal sequence was determined to be Gly-Glu-Tyr-Trp-Gln-Ser-Phe-. The enzyme is active on chitooligosaccharides with more than two N-acetylglucosamine residues (chitobiose) and is able to convert the nascent chitin synthesized by chitin synthase to chitosan in vitro. When O-hydroxyethylated chitin (glycol chitin) was used as a substrate, the optimum pH for enzyme activity was 5.0 and the optimum temperature was 50 degrees C. The enzyme was heat-stable and strongly inhibited by Fe3+. Furthermore, chitin deacetylase was demonstrated to be localized near the inner face of the cell wall (periplasmic space) in the mycelia by using immunoelectron microscopy.
TL;DR: The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+.
Abstract: Bacillus stearothermophilus T-6 produced an alpha-L-arabinofuranosidase when grown in the presence of L-arabinose, sugar beet arabinan, or oat spelt xylan. At the end of a fermentation, about 40% of the activity was extracellular, and enzyme activity in the cell-free supernatant could reach 25 U/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 20000, and the enzyme was purified eightfold by anion-exchange and hydrophobic interaction chromatographies. The molecular weight of T-6 alpha-L-arabinofuranosidase was 256,000, and it consisted of four identical subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The native enzyme had a pI of 6.5 and was most active at 70 degrees C and at pH 5.5 to 6.0. Its thermostability at pH 7.0 was characterized by half-lives of 53, 15, and 1 h at 60, 65, and 70 degrees C, respectively. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax, a Km, and an activation energy of 749 U/mg, 0.42 mM, and 16.6 kcal/mol, (ca. 69.5 kJ/mol), respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+. T-6 alpha-L-arabinofuranosidase released L-arabinose from arabinan and had low activity on oat spelt xylan. The enzyme acted cooperatively with T-6 xylanase in hydrolyzing oat spelt xylan, and L-arabinose, xylose, and xylobiose were detected as the end reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The understanding of the mechanism by which NTBC perturbs tyrosine catabolism has led to the clinical use of this chemical as the first effective pharmacological therapy for the hereditary disorder tyrosinemia I.
TL;DR: Enzyme activity was stable even after prolonged storage of the cell extract or of the purified protein under air, however, enzyme activity could be measured only in the presence of a strong reducing agent such as titanium(III) citrate or dithionite.
Abstract: Acetylene hydratase of the mesophilic fermenting bacterium Pelobacter acetylenicus catalyzes the hydration of acetylene to acetaldehyde. Growth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, molybdate supply in the medium. The specific enzyme activity in cell extract was highest after growth in the presence of tungstate. Enzyme activity was stable even after prolonged storage of the cell extract or of the purified protein under air. However, enzyme activity could be measured only in the presence of a strong reducing agent such as titanium(III) citrate or dithionite. The enzyme was purified 240-fold by ammonium sulfate precipitation, anion-exchange chromatography, size exclusion chromatography, and a second anion-exchange chromatography step, with a yield of 36%. The protein was a monomer with an apparent molecular mass of 73 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was at pH 4.2. Per mol of enzyme, 4.8 mol of iron, 3.9 mol of acid-labile sulfur, and 0.4 mol of tungsten, but no molybdenum, were detected. The Km for acetylene as assayed in a coupled photometric test with yeast alcohol dehydrogenase and NADH was 14 microM, and the Vmax was 69 mumol.min-1.mg of protein-1. The optimum temperature for activity was 50 degrees C, and the apparent pH optimum was 6.0 to 6.5. The N-terminal amino acid sequence gave no indication of resemblance to any enzyme protein described so far.
TL;DR: The partially purified enzyme showed a broad substrate specificity and hydroxylated the three isomeric cresols, chlorophenols and methylated chlorophenol, and was found to be essential for maximum activity of the cytoplasmically localized enzyme.
Abstract: Acinetobacter calcoaceticus NCIB 8250 utilizes phenol as sole source of carbon and energy via an ortho-cleavage pathway. The presence of ethanol in mixed substrate cultivations repressed the utilization of phenol. In fed batch cultivation the phenol tolerance was increased at least 2-fold. Maximum degradation rates of 150 mg phenol/(1 h) and 280 mg phenol/(g h), respectively were observed. Phenol hydroxylase is induced by its substrate and in parallel the catechol-1,2-dioxygenase is detectable. The presence of active phenol hydroxylase is strongly connected with the phenol degradation. Using a spectrophotometric enzyme assay the partially purified phenol hydroxylase was characterized with respect to kinetic parameters. The apparent Km values for phenol, FAD and NADPH were estimated to be 147 microM, 35 microM and 416 microM, respectively. Both FAD and NADPH were essential for maximum activity of the cytoplasmically localized enzyme. No substrate inhibition of phenol hydroxylase by phenol was observed up to 0.8 mM. The pH and temperature optima were pH 7.8 and 33 degrees C, respectively. The partially purified enzyme showed a broad substrate specificity. It hydroxylated the three isomeric cresols, chlorophenols and methylated chlorophenols. Pyrogallol, 3,4-dihydroxy-L-phenylalanine and resorcinol were oxygenated with higher rates than phenol. With the exception of phenol all other enzyme substrates tested did not serve as growth substrates.
TL;DR: Results showed that the enzyme formed exclusively [beta](2->6) fructosyl-fructose linkages, either initiating or elongating a fructan chain of the phlein type, and it is proposed to rename the purified enzyme sucrose:fructan 6-fructsyltransferase.
Abstract: Sucrose:sucrose 6-fructosyltransferase, an enzyme activity recently identified in fructan-accumulating barley (Hordeum vulgare) leaves, was further characterized. The purified enzyme catalyzed the transfer of a fructosyl group from sucrose to various acceptors. It displayed some [beta]-fructosidase (invertase) activity, indicating that water could act as fructosyl acceptor. Moreover, it transferred the fructosyl residue of unlabeled sucrose to [U-14C]Glc, producing [U-14C]sucrose and unlabeled glucose. Most significantly for fructan synthesis, the enzyme used as acceptors but not as donors a variety of oligofructans containing [beta](2->1)- and [beta](2->6)-linked fructosyl moieties. Thus, it acted as a general sucrose:fructan fructosyltransferase. The products formed by the enzyme from sucrose and various purified, structurally characterized oligofructans were analyzed by liquid chromatography and identified by comparison with structurally characterized standards. The results showed that the enzyme formed exclusively [beta](2->6) fructosyl-fructose linkages, either initiating or elongating a fructan chain of the phlein type. We propose, therefore, to rename the purified enzyme sucrose:fructan 6-fructosyltransferase.