scholarly journals Elicitor-induced prolyl hydroxylase from French bean (Phaseolus vulgaris). Localization, purification and properties

1985 ◽  
Vol 229 (3) ◽  
pp. 693-699 ◽  
Author(s):  
G P Bolwell ◽  
M P Robbins ◽  
R A Dixon
Keyword(s):  
Phaseolus Vulgaris ◽  
Cultured Cells ◽  
Smooth Endoplasmic Reticulum ◽  
Sodium Dodecyl ◽  
French Bean ◽  
Exchange Chromatography ◽  
Prolyl Hydroxylase ◽  
Colletotrichum Lindemuthianum ◽  
Phytopathogenic Fungus ◽  
Kinetic Properties

The enzyme prolyl hydroxylase (proline: 2-oxoglutarate dioxygenase, EC 1.14.11.12), induced in suspension-cultured cells of Phaseolus vulgaris L. (French bean) by treatment with an elicitor preparation from the phytopathogenic fungus Colletotrichum lindemuthianum, has been investigated. The enzyme, which catalyses the hydroxylation of poly-L-proline with the stoichiometric decarboxylation of 2-oxoglutarate, has been shown to be localized mainly in smooth endoplasmic reticulum. After solubilization from microsomal membranes, the hydroxylase was purified by ion-exchange chromatography and affinity chromatography on poly-L-proline-Sepharose 4B. The subunit Mr, as assessed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, was 65 000, the subunit apparently being recovered as a doublet: the subunits associate under non-denaturing conditions to give at least a tetramer. The bean hydroxylase has kinetic properties and cofactor requirements similar to those previously reported for the enzyme from other plants. Elicitor treatment of suspension-cultured bean cells leads to a rapid induction of prolyl hydroxylase activity concomitant with induction of a protein: arabinosyl-transferase and increased levels of an arabinosylated hydroxyproline-rich protein.

scholarly journals Differential patterns of arabinosylation by membranes of suspension-cultured cells of Phaseolus vulgaris (French bean) after subculture or elicitation

1984 ◽  
Vol 222 (2) ◽  
pp. 427-435 ◽  
Author(s):  
G P Bolwell
Keyword(s):  
Phaseolus Vulgaris ◽  
Cultured Cells ◽  
Initial Period ◽  
French Bean ◽  
Colletotrichum Lindemuthianum ◽  
Fungal Elicitor ◽  
Arabinogalactan Protein ◽  
Suspension Cultured Cells

Suspension-cultured cells of Phaseolus vulgaris (French bean) incorporated [1-3H] arabinose in vivo into high-Mr polymers that could be separated into glycoprotein and polysaccharide. Microsomal membranes from suspension-cultured cells of beans incorporated arabinose from UDP-beta-L-arabinose in vitro into both polysaccharide and glycoprotein. The enzyme involved in arabinan synthesis, arabinan synthase, appeared to be immunologically distinct from the protein:arabinosyltransferase system. Both these activities are inducible, but behave differently with either plant-growth-regulator or fungal-elicitor treatments. After subculture of cells entering the stationary growth phase the arabinan synthase activity reaches much higher values than does that of the protein transferase system during the initial period of cell division and growth, whereas after elicitation at the same growth stage, all the increased incorporation of arabinose occurs into glycoprotein of Mr higher than 200 000 and to a greater extent into a specific glycoprotein of Mr 42 500. Preliminary characterization of these glycoproteins prepared under non-reducing conditions and after acid and alkaline hydrolysis suggests that the high-Mr glycoprotein material is similar to arabinogalactan protein, whereas the lower-Mr material may be a hydroxyproline-rich protein existing as a dimer and that specifically increases during the hypersensitive response of the cells to the fungal elicitor from Colletotrichum lindemuthianum.

scholarly journals Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cell-wall peroxidase from French bean (Phaseolus vulgaris L.)

1994 ◽  
Vol 299 (3) ◽  
pp. 747-753 ◽  
Author(s):  
A Zimmerlin ◽  
P Wojtaszek ◽  
G P Bolwell
Keyword(s):  
Cell Wall ◽  
Phaseolus Vulgaris ◽  
Ferulic Acid ◽  
Cultured Cells ◽  
Gel Filtration ◽  
High Specificity ◽  
French Bean ◽  
Good Evidence ◽  
Ionization Mass ◽  
Phaseolus Vulgaris L

A cationic (pI 8.3) wall-bound peroxidase has been purified to homogeneity from suspension-cultured cells of French bean (Phaseolus vulgaris L.). The enzyme was a glycoprotein and its M(r) was 46,000 as determined by SDS/Page and h.p.l.c. gel filtration. It was localized biochemically to microsomes and the cell wall, and the latter subcellular distribution was confirmed by immunogold techniques. The native enzyme showed absorption maxima at 403, 500 and 640 nm, with an RZ (A405/A280) of 3.3. The peroxidase oxidized guaïacol and natural phenolic acids. By desorption-chemical-ionization mass spectrometry the enzyme was found to oxidize the model compound, ferulic acid, into dehydrodiferulic acid. Kinetics studies indicated an apparent Km of 113.3 +/- 22.9 microM and a Vmax of 144 mumol.min-1.nmol-1 of protein at an H2O2 concentration of 100 microM. In comparison with a second French-bean peroxidase (FBP) and horseradish peroxidase, as a model, it acted with a 6-10-fold higher specificity in this capacity. It is a member of the peroxidase superfamily of bacterial, fungal and plant haem proteins by virtue of its highly conserved amino acid sequence within the proximal and distal haem-binding sites. This is good evidence that this particular FBP may function in constructing covalent cross-linkages in the wall during development and response to pathogens.

scholarly journals Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1973 ◽  
Vol 133 (2) ◽  
pp. 335-347 ◽  
Author(s):  
J. C. Gray ◽  
R. G. O. Kekwick
Keyword(s):  
Phaseolus Vulgaris ◽  
Competitive Inhibition ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
French Bean ◽  
Exchange Chromatography ◽  
Mevalonate Kinase ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Green Leaves

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a Km of 4.26×10−5m for RS-mevalonate and 1.54×10−3m for ATP. The preparation from green leaves had a Km of 4.55×10−5m for RS-mevalonate and 1.75×10−3m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.

Response of Phaseolus vulgaris protoplasts to chemical components of fungal origin

1987 ◽  
Vol 65 (1) ◽  
pp. 63-68 ◽  
Author(s):  
Helen M. Griffiths ◽  
Anne J. Anderson
Keyword(s):  
Phaseolus Vulgaris ◽  
French Bean ◽  
Colletotrichum Lindemuthianum ◽  
Chemical Components ◽  
Low Concentrations ◽  
Extracellular Products ◽  
Bean Hypocotyl ◽  
Whole Plants ◽  
Hypocotyl Tissue ◽  
Plant Pathogen Interactions

Colletotrichum lindemuthianum is the causal agent of anthracnose in Phaseolus vulgaris L. (French bean). The α and β races of the fungus were used in this study with French bean cultivar ‘Great Northern’. Whole plants inoculated with the α race developed brown lesions on the hypocotyls (susceptible response). The β race caused small limited lesions, indicating a more resistant interaction. Extracellular products and cell wall materials were isolated from β race cultures and extracellular products from α race cultures. The extracts were size fractionated. By using a cotyledon bioassay, elicitor activity was demonstrated on ‘Dark Red Kidney’ within fractions from the α race. Fractions from the β race had little activity on ‘Dark Red Kidney’ or ‘Great Northern’ cotyledons. Protoplasts were isolated from ‘Great Northern’ bean hypocotyl tissue and incubated with the fungal fractions. Even at low concentrations (0.01 μg glucose equivalent∙mL−1), the β race culture filtrate rapidly killed a greater percentage of protoplasts (30%) than the α race (15%). The β race wall extract had little effect upon protoplast viability. The proportion of nonviable protoplasts depended on the incubation period and the concentration of the fungal material. Heat treatment of the culture filtrates and wall extract did not decrease their lethal effects. The results suggest that protoplasts may be valuable in examining the nature of certain plant–pathogen interactions.

scholarly journals l-phenylalanine ammonia-lyase from French bean (Phaseolus vulgaris L.). Characterization and differential expression of antigenic multiple Mr forms

1991 ◽  
Vol 279 (1) ◽  
pp. 231-236 ◽  
Author(s):  
G P Bolwell ◽  
M W Rodgers
Keyword(s):  
Phaseolus Vulgaris ◽  
Phenylalanine Ammonia Lyase ◽  
Cultured Cells ◽  
Peptide Mapping ◽  
Cell Types ◽  
French Bean ◽  
Wound Response ◽  
Leaf Tissues ◽  
Specific Affinity

L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) purified from suspension-cultured cells of French bean (Phaseolus vulgaris) has been further characterized. A number of techniques, including use of an antiserum and affinity probes, have established that all the antigenic polypeptides represent polymorphic Mr forms of the enzyme. These peptides include an apparently higher-Mr (83,000) form which shows different kinetics of induction from the Mr-77000 forms that have been extensively characterized previously. The larger subunit appeared to be PAL by the following criteria: (a) binding to specific affinity and antibody matrices; (b) peptide mapping; (c) active-site labelling; and (d) amino acid composition. The increased Mr of the larger subunit was not completely attributable to glycosylation, although some sugar residues were detected in this Mr-83000 form but not in the other Mr forms. Mr-83000 subunits were also immunoprecipitated from translations in vitro of mRNA from cells that had been stressed for a long period. They were also detected in leaf tissues that were not yet undergoing an extensive wound response. This form of the enzyme may be constitutive and involved in the low-level accumulation of phenolics in most cell types. By contrast, the Mr-77000 forms of PAL were rapidly induced during elicitor action, wounding or cytokinin-induced xylogenesis as a key regulatory enzyme involved in the synthesis of phenolics under stress conditions or during differentiation.

Purification and partial characterization of bovine kidney aldehyde dehydrogenase able to oxidize retinal to retinoic acid

1996 ◽  
Vol 74 (5) ◽  
pp. 695-700 ◽  
Author(s):  
Pangala V. Bhat ◽  
Lyne Poissant ◽  
Xiao Ling Wang
Keyword(s):  
Retinoic Acid ◽  
Molecular Mass ◽  
Aldehyde Dehydrogenase ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Anion Exchange Column ◽  
Bovine Kidney ◽  
Low K

A NAD-dependent enzyme that catalyzes the oxidation of retinal to retinoic acid has been purified to homogeneity from bovine kidney. The procedures used in the purification included ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Affi-gel blue and chromatography on a Mono-Q anion-exchange column. On the Mono-Q column, the enzyme aldehyde dehydrogenase (ALDH) resolved into two activity peaks designated as ALDH1 and ALDH2. The enzymes ALDH1 and ALDH2 were purified about 114- and 65-fold, respectively. Gel filtration chromatography of the partially purified native enzyme on Sephacryl S-200 HR exhibited a molecular mass of about 108 kDa. Electrophoresis of the purified enzymes under nondenaturing conditions showed a single protein band. However, sodium dodecyl sulfate – polyacrylamide gel electrophorsis indicated three protein bands in the 55, 30, and 22 kDa molecular mass regions. Both enzymes exhibited a broad substrate specificity oxidizing a wide variety of aliphatic and aromatic aldehydes. The ALDH1 enzyme had a pI of 7.45 and exhibited a low Km (6.37 μM) for retinal, while the ALDH2 enzyme was found to have very low Km for acetaldehyde (0.98 μM). Based on its kinetic properties, it is suggested that the ALDH1 enzyme may be the primary enzyme for oxidizing retinal to retinoic acid in bovine kidney.Key words: aldehyde dehydrogenase, vitamin A, retinal oxidation, retinoic acid.

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