Spectrophotometric determination of copper and iron subsequent to the simultaneous extraction of bis(2,9-dimethyl-1,10-phenanthroline)copper(I) and bis[2,4,6-tri(2-pyridyl)-1,3,5-triazine]iron(II) into propylene carbonate

Stephens, B. G.; Felkel, Hugo L.; Spinelli, W. M.

doi:10.1021/ac60342a013

Cited by 48 publications

(19 citation statements)

References 16 publications

(5 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At intervals, 50-l samples were taken, and the reaction was stopped by adding each sample to 1 ml of 0.5 M Na 2 CO 3 . The amount of reducing sugars liberated was determined by using the neocuproin reagent (42). One unit of enzyme is defined as the amount that generates 1 mol of reducing sugar in 1 h. The assay was also performed using other substrates: lemon pectins with different degrees of methylation, apple pectin and sugar-beet pectin (from Copenhagen Pectin), and modified hairy regions of pectin (36).…”

Section: Methodsmentioning

confidence: 99%

PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

Hugouvieux‐Cotte‐Pattat

Shevchik

Nasser

2002

J Bacteriol

View full text Add to dashboard Cite

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-␣-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.The enterobacterium Erwinia chrysanthemi is a well-characterized phytopathogen which causes soft-rot disease of various plants. The typical soft-rot maceration of plant tissues involves the depolymerization of the pectin in plant cell walls. The cleavage of this polysaccharide, an ␣-1,4-galacturonan (or polygalacturonate), is accomplished by a variety of microbial or fungal enzymes produced, in particular, by plant pathogens and by saprophytic microorganisms. Variations in the structure of the galacturonan chain occur in the extent of esterifications by O-acetyl and methoxyl groups, as well as in some modifications, such as xylose substitution or insertion in the polysaccharide backbone of single 1,2-linked ␣-L-rhamnose (27,37,46). Two type...

show abstract

Section: Methodsmentioning

confidence: 99%

PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

Hugouvieux‐Cotte‐Pattat

Shevchik

Nasser

2002

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…polygalacturonic acid in 0.1 M sodium acetate, pH 4.2, at 30°C. Reducing end groups were routinely determined by the neocuproine method as described by Stephens et al (1974) or by a modified ferricyanide test (Robyt et al, 1973) in the kinetic experiments, both with Dgalacturonate as a reference. One enzyme unit was defined as the amount of enzyme which produces 1 pmol reducing sugar/ min.…”

Section: Methodsmentioning

confidence: 99%

Primary Structure and Characterization of an Exopolygalacturonase from Aspergillus Tubingensis

Kester

Someren

Muller

et al. 1996

European Journal of Biochemistry

View full text Add to dashboard Cite

From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly( 1,4-a-~-ga~acturonide)galacturonohydrolasel that cleaves monomer units from the non-reducing end of the substrate molecule.K,,, and V,,,, for polygalacturonic acid hydrolysis were 3.2 mg mlV' and 3.1 mg ml-' and 255 U mg I and 262U mg-' for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2 -7), the Michaelis constant (K,,,) decreased with increasing chain length (n). The V,;,, value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid ( K , = 0.3 mM) as well as by reduced digalacturonate (K, = 0.4 mM).The exopolygalacturonase gene (pgax) was cloned by reverse genetics and shows only 13 % overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pguX-coding region was used to achieve high level production of exopol ygalacturonase under conditions where no endopolygalacturonases were produced.

show abstract

“…The assay buffer was equilibrated at 30°C, and the reaction was initiated by the addition of 20 l of enzyme solution in the same buffer at a concentration with which the reaction rates were linear over the selected time course. The amount of reducing end groups liberated after different incubation times was measured as described (19). In addition, the polygalacturonase activity was also measured in 20 mM methyl-piperazine/HCl and McIlvaine buffers (pH 4.2) (20) to study the ionic strength dependence of each of the mutated forms of PGII.…”

mentioning

confidence: 99%

The Active Site Topology of Aspergillus nigerEndopolygalacturonase II as Studied by Site-directed Mutagenesis

Armand¹,

Wagemaker²,

Sánchez-Torres³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K m and V max ) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta-and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.Pectic polysaccharides are among the most complex plant cell wall polysaccharides. In the homogalacturonan part, the so-called smooth regions, the 1,4-␣-D-galacturonic acid backbone is partly esterified. These smooth regions are interspersed by the rhamnogalacturonan parts consisting of repeating stretches of 1,2-␣-L-rhamnose-1,4-␣-D-galacturonic acid dimers. Other sugar residues can be attached to the rhamnose residues (1). Because of this complexity, a wide range of enzymes, the so-called pectinases, is necessary for the complete degradation of pectic substances. Two main classes of depolymerizing enzymes act on these polysaccharides: the hydrolases (endopolygalacturonases and rhamnogalacturonases) and the lyases (pectin lyase, pectate lyase, and rhamnogalacturonan lyase).Endopolygalacturonases (PGs; EC 3.2.1.15) 1 catalyze the random hydrolysis of 1,4-␣-D-galactosiduronic linkages in pectates. They have been isolated from a variety of organisms (eukaryotae and prokaryotae). Furthermore, over 40 genes encoding PGs have been cloned and sequenced. The corresponding enzymes have been grouped in family 28 of the general classification of glycosyl hydrolases based on amino acid sequence similarities (2, 3).The gene encoding the endopolygalacturonase II (PGII) from Aspergillus niger has been previously cloned, sequenced, and expressed in A. niger (4). The enzyme hydrolyses the glycosidic linkages with inversion of configuration (5). Recently, PGII was extensively characterized with respect to activity on polygalacturonic acid, mode of action, and kinetics on oligogalacturonates (6).Two different mechanisms have been identified for glycosyl hydrolases: one resulting in retention and the other in inversion of the configuration at the anomeric carbon of the scissile bond (7,8). Despite this difference, in most glycosidases two residues ...

show abstract

Spectrophotometric determination of copper and iron subsequent to the simultaneous extraction of bis(2,9-dimethyl-1,10-phenanthroline)copper(I) and bis[2,4,6-tri(2-pyridyl)-1,3,5-triazine]iron(II) into propylene carbonate

Cited by 48 publications

References 16 publications

PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

Primary Structure and Characterization of an Exopolygalacturonase from Aspergillus Tubingensis

The Active Site Topology of Aspergillus nigerEndopolygalacturonase II as Studied by Site-directed Mutagenesis

Contact Info

Product

Resources

About