Showing papers on "Enzyme assay published in 2005"
TL;DR: Results suggest that Al toxicity and water stress decrease total amount of functional NR in rice seedlings and the osmolytes proline, glycine betaine and sucrose appear to have a direct protective action on enzyme NR under stressful conditions.
287 citations
TL;DR: It is demonstrated that pretreated corn stover is a good substrate both for enzyme production and hydrolysis, since high cellulolytic activities could be reached using it as carbon source and high sugar yields could be obtained in the Hydrolysis by the enzyme produced on steam pretreatedcorn stover.
273 citations
TL;DR: In addition to its major application in animal nutrition, phytase is also used for processing of human food and research in this field focuses on better mineral absorption and technical improvement of food processing.
Abstract: Phytases decompose phytate, which is the primary storage form of phosphate in plants. More than 10 years ago, the first commercial phytase product became available on the market. It offered to help farmers reduce phosphorus excretion of monogastric animals by replacing inorganic phosphates by microbial phytase in the animal diet. Phytase application can reduce phosphorus excretion by up to 50%, a feat that would contribute significantly toward environmental protection. Furthermore, phytase supplementation leads to improved availability of minerals and trace elements. In addition to its major application in animal nutrition, phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing. All commercial phytase preparations contain microbial enzymes produced by fermentation. A wide variety of phytases were discovered and characterized in the last 10 years. Initial steps to produce phytase in transgenic plants were also undertaken. A crucial role for its commercial success relates to the formulation of the enzyme solution delivered from fermentation. For liquid enzyme products, a long shelf life is achieved by the addition of stabilizing agents. More comfortable for many customers is the use of dry enzyme preparations. Different formulation technologies are used to produce enzyme powders that retain enzyme activity, are stable in application, resistant against high temperatures, dust-free, and easy to handle.
270 citations
TL;DR: This pathway operates extracellularly in cultured Rosa cells, proceeds via several novel intermediates including 4-O-oxalyl-l-threonate, and involves at least one new enzyme activity, and can also operate non-enzymatically, potentially accounting for vitamin losses during cooking.
Abstract: Increasing the l-ascorbate (vitamin C) content of crops could in principle involve promoting its biosynthesis or inhibiting its degradation. Recent progress has revealed biosynthetic pathways for ascorbate1,2,3, but the degradative pathways remain unclear. The elucidation of such pathways could promote an understanding of the roles of ascorbate in plants4, and especially of the intriguing positive correlation between growth rate and ascorbate oxidase5,6 (or its products7). In some plants (Vitaceae), ascorbate is degraded via l-idonate to l-threarate (l-tartrate), with the latter arising from carbons 1–4 of ascorbate3,8,9,10,11. In most plants, however (including Vitaceae)11, ascorbate degradation can occur via dehydroascorbate, yielding oxalate12 plus l-threonate, with the latter from carbons 3–6 of ascorbate3,10,13. The metabolic steps between ascorbate and oxalate/l-threonate, and their subcellular location, were unknown. Here we show that this pathway operates extracellularly in cultured Rosa cells, proceeds via several novel intermediates including 4-O-oxalyl-l-threonate, and involves at least one new enzyme activity. The pathway can also operate non-enzymatically, potentially accounting for vitamin losses during cooking. Several steps in the pathway may generate peroxide; this may contribute to the role of ascorbate as a pro-oxidant14,15 that is potentially capable of loosening the plant cell wall and/or triggering an oxidative burst.
254 citations
TL;DR: A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized.
239 citations
TL;DR: Recent advances in understanding of the structure, mechanism, and regulation of cystathionine beta-synthase are discussed from the perspective of its physiological function, focusing on the clinically relevant human enzyme.
212 citations
TL;DR: Results show that post-translational regulatory mechanisms are likely to play an important role in modulating FAD2-1 enzyme activities.
Abstract: The endoplasmic reticulum-associated oleate desaturase FAD2 (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine Delta12-desaturase) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues of plants. Little is known, however, concerning the post-transcriptional mechanisms that regulate the activity of this important enzyme. The soybean genome possesses two seed-specific isoforms of FAD2, designated FAD2-1A and FAD2-1B, which differ at only 24 amino acid residues. Expression studies in yeast revealed that the FAD2-1A isoform is more unstable than FAD2-1B, particularly when cultures were maintained at elevated growth temperatures. Analysis of chimeric FAD2-1 constructs led to the identification of two domains that appear to be important in mediating the temperature-dependent instability of the FAD2-1A isoform. The enhanced degradation of FAD2-1A at high growth temperatures was partially abrogated by treating the cultures with the 26S proteasome-specific inhibitor MG132, and by expressing the FAD2-1A cDNA in yeast strains devoid of certain ubiquitin-conjugating activities, suggesting a role for ubiquitination and the 26S proteasome in protein turnover. In addition, phosphorylation state-specific antipeptide antibodies demonstrated that the Serine-185 of FAD2-1 sequences is phosphorylated during soybean seed development. Expression studies of phosphopeptide mimic mutations in yeast suggest that phosphorylation may downregulate enzyme activity. Collectively, the results show that post-translational regulatory mechanisms are likely to play an important role in modulating FAD2-1 enzyme activities.
186 citations
TL;DR: High‐performance liquid chromatography and mass spectrometry analyses revealed that mammalian sera had a lactonase‐like enzyme, which hydrolyzed the lactone ring of AHL to produce acyl homoserine, with enzyme properties reminiscent of paraoxonases (PONs).
184 citations
TL;DR: The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme, and the highest activity was observed with branched-chain α-keto acids, indicating that the enzyme might be active under cheese ripening conditions.
Abstract: The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA) The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains By using a random mutagenesis approach, the gene encoding KdcA in L lactis B1157 was identified The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L lactis IL1403 Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase The kdcA gene was overexpressed in L lactis for further characterization of the decarboxylase enzyme Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 63, indicating that the enzyme might be active under cheese ripening conditions
175 citations
TL;DR: The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.
Abstract: Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.
174 citations
TL;DR: Both ACE2 and ACE are ectoenzymes that have distinct localization and secretion patterns that determine their role on the cell surface in kidney epithelium and in urine.
TL;DR: It is hypothesized that GSPE probably improves the cellular redox status via glutathione synthesis pathways instead of regulation of the GPx and/or GR activities protecting against oxidative damage.
Abstract: In the present paper, we report the effect of a grape seed procyanidin extract (GSPE) on antioxidant enzyme systems (AOEs). Gene expression was tested using the hepatocarcinoma cell line HepG2 by exposing it to several GSPE doses between 0 and 100 mg/L for 24 h. We evaluated mRNA expression and enzyme activity levels using real time RT-PCR and spectrophotometry. The results suggested a transcriptional GSPE regulation of glutathione related enzymes caused by an increase both in mRNA and in enzyme activity levels overall at 15 mg/L. We also assessed the GSPE effect on AOEs in cells submitted to oxidative stress. Under oxidative conditions (1 mM H(2)O(2), 1 h), we found a decrease in GSH content and an increase in MDA, and we suggested a posttranslational regulation of GPx/GR mRNAs and a transcriptional enhancement of GST mRNA. The GSPE pretreatment (15 mg/L, 23 h) before HepG2 submission to H(2)O(2) (1 mM, 1 h) showed an increase of the mRNA of GPx/GR with respect to the H(2)O(2) group, whereas the GSH content was similar to the control group. However, the GPx/GR enzyme activities were not increased. We hypothesize that GSPE probably improves the cellular redox status via glutathione synthesis pathways instead of regulation of the GPx and/or GR activities protecting against oxidative damage.
TL;DR: The enzyme inhibition of 73 and 93% by pepstatin at 10 and 20 μM, respectively proved it to be an aspartate protease; however, the additional requirement of histidine residue for enzyme activity has been indicated by differential spectra of diethyl pyrocarbonate treated versus untreated enzyme.
TL;DR: A role for H2S in carrageenan‐induced hindpaw oedema in the rat is identified and a dose‐dependent manner is identified.
Abstract: Hydrogen sulphide (H2S) is a naturally occurring gas, with potent vasodilator activity. In this report, we identify a role for H2S in carrageenan-induced hindpaw oedema in the rat. Intraplantar injection of carrageenan (150 μl, 2% (w v−1)) resulted in an increase in hindpaw H2S synthesising enzyme activity and increased myeloperoxidase (MPO) activity. Pretreatment (i.p. 60 min before carrageenan) with DL-propargylglycine (PAG, 25–75 mg kg−1), an inhibitor of the H2S synthesising enzyme cystathionine-γ-lyase (CSE), significantly reduced carrageenan-induced hindpaw oedema in a dose-dependent manner (e.g. increase in hindpaw weight at 3 h, saline: 0.12±0.017 g; carrageenan, 1.39±0.037 g; PAG, 50 mg kg−1, 1.11±0.06 g, n=10) and MPO activity (fold increase) in the hindpaw (saline: 1.0±0.12; carrageenan, 2.92±0.45 g; PAG, 50 mg kg−1, 1.1±0.22, n=10); PAG (50 mg kg−1) also inhibited H2S synthesising enzyme activity (nmol μg DNA−1) in the hindpaw in a dose-dependent manner (saline, 0.46±0.05; carrageenan, 0.71±0.08 g; PAG, 50 mg kg−1, 0.17±0.05, n=10).
British Journal of Pharmacology (2005) 145, 141–144. doi:10.1038/sj.bjp.0706186
TL;DR: Lesion size was significantly reduced in transgenic plants compared to nontransformed controls, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.
Abstract: Sclerotinia minor Jagger is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Based on evidence that oxalic acid is involved in the pathogenicity of many Sclerotinia species, our objectives were to recover transgenic peanut plants expressing an oxalic acid-degrading oxalate oxidase and to evaluate them for increased resistance to S. minor. Transformed plants were regenerated from embryogenic cultures of three Virginia peanut cultivars (Wilson, Perry, and NC-7). A colorimetric enzyme assay was used to screen for oxalate oxidase activity in leaf tissue. Candidate plants with a range of expression levels were chosen for further analysis. Integration of the transgene was confirmed by Southern-blot analysis, and gene expression was demonstrated in transformants by northern-blot analysis. A sensitive fluorescent enzyme assay was used to quantify expression levels for comparison to the colorimetric protocol. A detached leaflet assay tested whether transgene expression could limit lesion size resulting from direct application of oxalic acid. Lesion size was significantly reduced in transgenic plants compared to nontransformed controls (65%-89% reduction at high oxalic acid concentrations). A second bioassay examined lesion size after inoculation of leaflets with S. minor mycelia. Lesion size was reduced by 75% to 97% in transformed plants, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.
TL;DR: Analysis of 83 families containing two parents and two children demonstrated significant correlations between basal activity in parent-child pairs and sibling pairs, but not spousal pairs, suggesting strong genetic control of basal activity.
Abstract: It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine triphosphate in 2'-specific nucleotidyl transfer reactions to synthesize 2',5'-oligoadenylates, which activate latent ribonuclease, resulting in degradation of viral RNA and inhibition of virus replication. We showed elsewhere that constitutive (basal) activity of 2'5'AS is correlated with virus-stimulated activity. In the present study, we asked whether constitutive activity is genetically determined and, if so, by which variants. Analysis of 83 families containing two parents and two children demonstrated significant correlations between basal activity in parent-child pairs (P<.0001) and sibling pairs (P=.0044), but not spousal pairs, suggesting strong genetic control of basal activity. We next analyzed association between basal activity and 15 markers across the OAS gene cluster. Significant association was detected at multiple markers, the strongest being at an A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (AG or AA) of the OAS1 gene. At this unusual polymorphism, allele G had a higher gene frequency in persons with high enzyme activity than in those with low enzyme activity (0.44 vs. 0.20; P=3 x 10(-11)). Enzyme activity varied in a dose-dependent manner across the GG, GA, and AA genotypes (tested by analysis of variance; P=1 x 10(-14)). Allele G generates the previously described p46 enzyme isoform, whereas allele A ablates the splice site and generates a dual-function antiviral/proapoptotic p48 isoform and a novel p52 isoform. This genetic polymorphism makes OAS1 an excellent candidate for a human gene that influences host susceptibility to viral infection.
TL;DR: Immobilized peroxidases from Momordica charantia were highly effective in decolorizing reactive textile dyes compared to its soluble counterpart and was capable of removing remarkably high concentration of color from the effluent.
TL;DR: It is evident that Achatina fulica can be used as a bioindicator to monitor the environmental heavy metal pollution.
Abstract: Heavy metal stress results in the production of O(2)(.-), H(2)O(2) and (.)OH, which affect various cellular processes, mostly the functioning of membrane systems. Cells are normally protected against free oxyradicals by the operation of intricate antioxidant systems. The aim of the present work is to examine the effect of CdCl(2) and ZnSO(4) on antioxidative enzyme activity in the gastropod, Achatina fulica. The concentrations of antioxidant enzymes--superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GPx)--and nonenzymatic antioxidants--glutathione and vitamin-C--were found to be decreased in both digestive gland and kidney of the gastropod, Achatina fulica treated with individual concentrations of 0.5 ppm and 1ppm of CdCl(2) and ZnSO(4), compared to that of control animals. Based on the above study, it is evident that Achatina fulica can be used as a bioindicator to monitor the environmental heavy metal pollution.
TL;DR: Bacillus sp.
01 Jan 2005
TL;DR: Out of 115 fungal cultures isolated from environmental sources including soil, air and infected plant, 78 (67.83%) were found to possess cellulose degrading ability, and optimal condition for enzyme activity and induction of enzyme synthesis were determined.
Abstract: Cellulases are a group of hydrolytic enzymes capable of hydrolyzing the most abundant organic polymer i.e. cellulose to smaller sugar components including glucose subunits. Cellulases have enormous potential in industries and are used in food, beverages, textile, laundry, paper and pulp industries etc. This study was aimed to screen the cellulytic ability of fungi from native environmental source. Furthermore, optimal condition for enzyme activity and induction of enzyme synthesis were also determined. Out of 115 fungal cultures isolated from environmental sources including soil, air and infected plant, 78 (67.83%) were found to possess cellulose degrading ability. Cellulytic fungi belonged to Aspergillus sp., Trichoderma sp., Fusarium sp., Alternaria sp., Penicillium sp., and Rhizopus sp. Cellulase production by fungi was measured by using enzyme assays. Highest yield of enzyme was noted at 37°C while maximum activity in the range of pH 4 4.8. Kinetics of enzyme production was also studied on selected Aspergillus isolates and mostly high enzyme production was observed after 7 days. Cellulase synthesis increased by ~10 folds in the presence of cellulose while it repressed in the presence of glucose.
Journal Article•
TL;DR: The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.
Abstract: Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.
TL;DR: High molecular weight enzymes, such as bacterial β-galactosidase, can be conjugated to BBB targeting antibodies for effective delivery across the blood-brain barrier in vivo.
Abstract: Enzyme replacement therapy of lysosomal storage disorders is complicated by the lack of enzyme transport across the blood-brain barrier (BBB). The present studies evaluate the delivery of a model enzyme across the BBB following enzyme conjugation to a BBB receptor-specific monoclonal antibody (mAb). Bacterial β-galactosidase (116 kDa) was conjugated to the rat 8D3 mAb to the rat transferrin receptor (TfR) via a streptavidin-biotin linkage. The unconjugated β-galactosidase or the β-galactosidase-8D3 conjugate was injected intravenously in adult mice, and enzyme activity was measured at 1 and 4 h in brain and peripheral organs (liver, spleen, kidney, and heart). Unconjugated β-galactosidase was rapidly removed from the blood compartment owing to avid uptake by liver and spleen. There was minimal uptake of the unconjugated β-galactosidase by brain. Following conjugation of the enzyme to the 8D3 TfRmAb, there was a 10-fold increase in brain uptake of the enzyme based on measurement of enzyme activity. Histochemistry of brain showed localization of the enzyme in the intraendothelial compartment of brain following intravenous injection of the enzyme-mAb conjugate. The capillary depletion technique showed that more than 90% of the enzyme-8D3 conjugate that entered into the endothelial compartment of brain passed through the BBB to enter brain parenchyma. In conclusion, high molecular weight enzymes, such as bacterial β-galactosidase, can be conjugated to BBB targeting antibodies for effective delivery across the BBB in vivo. Fusion proteins comprised of BBB targeting antibodies and recombinant enzymes could be therapeutic in the treatment of the brain in human lysosomal storage disorders.
TL;DR: The heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica is developed as a first step for enzyme evolution.
Abstract: Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.
TL;DR: Both concentration of the metal and length of exposure were found to influence enzyme activity, with higher concentrations of metals assumed to induce stress proteins like metallothioneins.
TL;DR: The results show that—in addition to the wood-colonizing white-rot fungi—the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.
Abstract: The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l−1) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l−1) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the “blue copper oxidases”. The enzyme’s pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20°C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40°C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (Km, kcat): 63 μM, 21 s−1 for ABTS, 4 μM, 5 s−1 for syringaldazine, 1,026 μM, 15 s−1 for DMP and 4307 μM, 159 s−1 for guaiacol. The results show that—in addition to the wood-colonizing white-rot fungi—the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.
TL;DR: For a given rate of enzyme synthesis, induction of the activities of phenylalanine ammonia-lyase and chalcone synthase is more efficient at high elicitor concentrations, which may reflect the operation under certain circumstances of post-translational control of the activity levels of these enzymes.
Abstract: Changes in the rate of synthesis of phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, have been investigated by direct immunoprecipitation of in vivo [35S]methionine-labelled enzyme subunits in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris). Elicitor, heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked but transient increases in the rates of synthesis of both enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased rates of synthesis of both enzymes can be observed 20 min after elicitor treatment and the pattern of induction of synthesis of phenylalanine ammonia-lyase and chalcone synthase are broadly similar with respect to elicitor concentration and time, maximum rates of synthesis being attained between 2.5 h and 3.0 h after elicitor treatment. Within this overall co-ordination small but distinct differences between the enzymes were observed in: (a) the elicitor concentrations giving maximum enzyme synthesis, and (b) the precise timing of maximum enzyme synthesis, with that for chalcone synthase occurring 20-30 min earlier than that for phenylalanine ammonia-lyase. However, for a given rate of enzyme synthesis, induction of the activities of phenylalanine ammonia-lyase and chalcone synthase is more efficient at high elicitor concentrations. This may reflect the operation under certain circumstances of post-translational control of the activity levels of these enzymes as implicated for phenylalanine ammonia-lyase by previous density-labelling experiments [Lawton et al. (1980) Biochim. Biophys. Acta, 633, 162-175]. The same pattern of induction of enzyme synthesis is observed with elicitor preparations from a variety of sources.
TL;DR: Sabar et al. as mentioned in this paper used a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of protein complexes, and in-gel histochemical staining of the enzyme activities.
Abstract: Our knowledge of the respiratory chain and associated defects depends on the study of the multisubunit protein complexes in the inner mitochondrial membrane. Functional analysis of the plant mitochondrial respiratory chain has been successfully achieved by a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of the protein complexes, and in-gel histochemical staining of the enzyme activities. We have optimized this powerful technique by determining linear ranges of amount of protein and enzyme activity for each respiratory complex. Time courses of the in-gel enzyme activities were also performed to determine optimal reaction times. Using the in-gel activity staining method we have previously shown decreased activity of complex V (F(1)F(0)-ATPase) in male-sterile sunflowers (Sabar et al., 2003). Here we have identified unique supercomplexes comprising complex IV (cytochrome c oxidase) in sunflower mitochondria. This method therefore represents a reliable tool for the diagnosis of respiratory dysfunction. In addition, the wider application of BN-PAGE in combination with enzyme activity staining is discussed.
TL;DR: In this paper, a plant UGAT called BpUGAT was found to catalyze the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2''-hydroxyl group of the 3-oglucosyl moiety of cyanidin 3-O-6''-Omalonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C.
Journal Article•
TL;DR: A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456, and the activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+.
Abstract: A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 degrees C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61%; higher activity than NADH. The Km values for NADPH and NADH were determined to be 47.5 and 17.2 micromol, and the Vmax values 322.2 and 130.7 micromol Cr(VI) min(-1)mg(-1) protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.