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Biomedical subjects

A M Boudet

Publications and source records attributed to A M Boudet.

At least 37 records · Page 2Linked to original sources
PubMed1993

Molecular cloning and expression of a Eucalyptus gunnii cDNA clone encoding cinnamyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the reduction of hydroxycinnamyl aldehydes (sinapyl, paracoumaryl, coniferyl aldehydes) to the corresponding alcohols which are the direct monomeric precursors of lignins. Recently, we have purified from Eucalyptus gunnii two isoforms of CAD (CAD1 and CAD2), distinct in their biochemical and functional properties. In this paper, we report the cloning of a CAD cDNA (pEuCAD2) isolated by screening a lambda gt11 library generated from cell suspension culture of Eucalyptus gunnii, using a tobacco CAD cDNA as a probe. This full-length clone (1392 bp) encodes a protein of 356 amino acids which corresponds to the subunit molecular weight of the CAD2 isoform. Sequence analysis revealed that CAD2 is very well conserved among species (78% homology with CAD from tobacco, a herbaceous angiosperm, and 81% with the partial sequence from a gymnosperm, loblolly pine). The identity of this clone was unambiguously demonstrated (1) by comparison with peptide sequence data from purified CAD2 and (2) by functional expression of the recombinant enzyme in Escherichia coli. Recombinant CAD showed the same properties as the natural isoform CAD2, in terms of electrophoretic mobility, polypeptide structure, substrate specificity and antigenicity. The CAD2 transcript is equally abundant in stems and leaves and at the limit of detection in roots. At the tissue level the CAD2 gene is highly expressed in xylem and virtually undetectable in phloem.

Alcohol Oxidoreductases↗
PubMed1993

Characterization of chilling-acclimation-related proteins in soybean and identification of one as a member of the heat shock protein (HSP 70) family.

Through a 5-d exposure at 14 degrees C/8 degrees C (day/night), soybean (Glycine max [L.] Merr.) was acclimated to a lower temperature of 8 degrees C. In order to assess changes in protein synthesis related to chilling acclimation, proteins were labeled in vivo with [35S]methionine, separated by two-dimensional gel electrophoresis, and the derived autoradiograms were subjected to computer analysis. Two sets of chilling-acclimation-related proteins were characterized following exposure and labeling at 8 degrees C. One set corresponded to proteins whose synthesis was stimulated in acclimated plants in comparison with non-acclimated plants after transfer to 8 degrees C for 2 d. The other set also displayed an enhanced synthesis in the acclimated plants versus the non-acclimated plants but after 7 d of exposure at 8 degrees C. Most of these chilling-acclimation-related proteins were not increased during the acclimation period at 14 degrees C. Using microsequence analysis, one of these proteins was shown to have a high sequence homology with members of the heat-shock protein (HSP 70) family.

Acclimatization↗
PubMed1992

Lateral and Rotational Mobilities of Lipids in Specific Cellular Membranes of Eucalyptus gunnii Cultivars Exhibiting Different Freezing Tolerance.

Two cell lines of Eucalyptus gunnii have been shown to keep their differential frost tolerance at the cellular level after long-term culture. They have been used to investigate the fluidity of specific cell membranes in relation with frost tolerance. Protoplasts and isolated vacuoles were obtained from both cell lines. In addition, purified plasma membrane and tonoplast (the vacuolar membrane) were separated from a crude microsomal fraction through free-flow electrophoresis. The lateral and rotational mobilities of lipids in these different membranes were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarization. After labeling the vacuoles isolated from the frost-sensitive cells with 1-oleoyl-2-(7-nitro-2,1,3-benz-oxadiazol-4-yl)aminocaproyl phosphatidylcholine, a single mobile component was observed with a diffusion coefficient of 2.4 x 10(-9) cm(2) s(-1) and a mobile fraction close to 100% at a temperature of 23 degrees C. When using isolated vacuoles from the frost tolerant line, a higher lateral diffusion of tonoplast lipids was found with a diffusion coefficient of 3.2 x 10(-9) cm(2) s(-1), still with a mobile fraction close to 100%. No convincing data were obtained when performing fluorescence recovery after photobleaching experiments on protoplasts. Fluorescence polarization experiments confirmed the differential behavior of the two cell lines for tonoplast and also for plasma membrane. In addition, they showed that intrinsically tonoplast exhibited a higher fluidity than plasma membrane. Our results provide the first information on the fluidity of tonoplast and on the compared properties of two important plant membranes-tonoplast and plasma membrane-through the use of two complementary biophysical approaches. In addition, they suggest there is a correlation between membrane fluidity and cold tolerance. The potential interest of plant vacuole as a natural model system in membrane studies is emphasized.

Journal Article↗
PubMed1992

Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H(+) extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O(2) uptake with a 4H(+) to O(2) ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.

Journal Article↗
PubMed1990

Tonoplast vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis.

Tonoplast vesicles were purified from a microsomal fraction isolated from etiolated soybean hypocotyls (Glycine max L.) by preparative free-flow electrophoresis. Marker enzyme determinations and immunoblot analysis against the vacuolar-ATPase confirmed the nature and the purity of the isolated membranes. A purified tonoplast fraction also was obtained by consecutive sucrose and glycerol centrifugation which was further resolved into two different populations of vesicles (T(A) and T(B)) by free-flow electrophoresis. The determination of the sidedness of these different vesicles included concanavalin A binding as an imposed label, NADH-ferricyanide oxidoreductase cytochemistry, and ATPase latency. The tonoplast fractions, obtained by consecutive sucrose and glycerol gradient centrifugations, were found to consist of a mixture of two populations of vesicles of opposite sidedness. The least electronegative fraction obtained by free-flow electrophoresis (T(B)) consisted predominantly of cytoplasmic side out tonoplast vesicles while a fraction of greater electronegativity (T(A)) contained the cytoplasmic side in tonoplast vesicles. The relative amounts of each type of vesicle varied with the method of homogenization. Razor blade chopping, Polytron, and Waring Blendor homogenization gave predominantly cytoplasmic side out vesicles, whereas mashing with a mortar and pestle gave nearly equal amounts of the two populations of membrane vesicles of different orientation.

Journal Article↗
PubMed1988

Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor.

Cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyzes the final step in a branch of phenylpropanoid synthesis specific for production of lignin monomers. We have isolated a full-length cDNA clone encoding CAD, as a molecular marker specific for lignification, by immunoscreening a lambda gt11 library containing cDNAs complementary to mRNA from elicitor-treated cell cultures of bean (Phaseolus vulgaris L.). The clone comprises a single long open reading frame of 1767 base pairs, 31 base pairs of 5' leader, and 152 base pairs of 3' untranslated sequence. The deduced 65-kDa CAD polypeptide has several features that are strongly conserved in alcohol dehydrogenases. Addition of fungal elicitor to cell cultures stimulates CAD transcription, which leads to a remarkably rapid, but transient, accumulation of CAD mRNA, with no detectable lag and maximal levels after 1.5 hr. Southern blot analysis of bean genomic DNA indicates that elicitor-induced CAD is encoded by a single gene. The regulatory significance of the rapid activation of this CAD gene and the possible existence of a second, divergent CAD gene involved in lignification during xylogenesis are discussed.

Alcohol Oxidoreductases↗
PubMed1988

Plasma membrane vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis.

Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction ;E,' was pure plasma membrane. The other, more electronegative and designated fraction ;C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.

Journal Article↗
PubMed1986

Rapid Degradation of Abnormal Proteins in Vacuoles from Acer pseudoplatanus L. Cells.

In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([(14)C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([(14)C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole.

Journal Article↗
PubMed1985

Hydrolysis of Intracellular Proteins in Vacuoles Isolated from Acer pseudoplatanus L. Cells.

Acer pseudoplatanus cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of [(3)H]leucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the [(3)H]leucine-labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20 degrees C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins.

Journal Article↗
PubMed1984

Purification and properties of cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase from poplar stems (Populus X euramericana).

Cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase were purified to apparent homogeneity from poplar stems (Populus euramericana) and their main properties were studied. Only one form was identified for each enzyme. The reductase corresponded to one polypeptide of molecular weight 36 000 and the cinnamyl alcohol dehydrogenase was constituted of two identical subunits of molecular weight 40 000. These characteristics are in agreement with most of the data obtained for the same enzymes isolated from other plants. The two reductive enzymes are inhibited by thiol reagents and a metal chelator 1,10-phenanthroline. The isoelectric point of the reductase (pH 7.5) and of the dehydrogenase (pH 5.6) were determined by chromatofocusing. The cinnamoyl-CoA reductase exhibit a decreasing affinity towards feruloyl-CoA, sinapoyl-CoA and p-coumaroyl-CoA. The cinnamyl alcohol dehydrogenase, which catalyses the reduction of the three cinnamaldehydes, exhibits its highest efficiency towards coniferaldehyde. In spite of differences in the monomeric composition of lignins from xylem and sclerenchyma the reductive enzymes isolated from these two lignified tissues exhibit the same substrate specificity. Consequently, they do not play an important role in the qualitative control of lignins in poplar tissues.

Alcohol Oxidoreductases↗
PubMed1983

Activation of plant quinate:NAD 3-oxidoreductase by Ca and calmodulin.

Quinate:NAD(+) 3-oxidoreductase (EC 1.1.1.24) from carrot cell suspension cultures has previously been shown to be activated by phosphorylation and inactivated by dephosphorylation. Here it is shown that the reactivation of the inactivated quinate:NAD(+) oxidoreductase is an enzyme-mediated process that requires ATP and protein kinase activity. The reactivation is completely inhibited by EGTA and can be restored by the addition of Ca(2+). Cyclic AMP at concentrations up to 5 muM did not have any effect on the reactivation either with or without EGTA in the medium. Calmodulin-depleted fractions containing quinate:NAD(+) oxidoreductase were obtained by passage of the crude extracts through an affinity column of 2-chloro-10-(3-aminopropyl)phenothiazine coupled to Sepharose 4B. The enzyme in this calmodulin-deficient fraction could be inactivated but not reactivated even in the presence of ATP and Ca(2+). However, addition of bovine brain calmodulin completely restored the activity of the enzyme. Half-maximal activation occurred at 130 nM calmodulin. We conclude from these data that the quinate:NAD(+) oxidoreductase is activated by a Ca(2+) - and calmodulin-dependent plant protein kinase.

Journal Article↗
PubMed1981

Isolation and Characterization of Vacuoles from Melilotus alba Mesophyll.

Methods for the preparation of protoplasts and vacuoles from mesophyll tissues of sweet clover (Melilotus alba Desr.) are described. Vacuoles are obtained using a new procedure which involves lysis of the plasmalemma during a brief centrifugation of protoplasts through a diethylaminoethyl dextran layer. This method combines the release of vacuoles and their purification in one step. The contamination of vacuole preparations was found to be low, as judged by enzymic markers and microscopic inspection. The method described is rapid and gives a good yield of vacuoles without causing changes in osmotic pressure. Several hydrolases were found to be located in vacuoles from sweet clover, which were also examined for their amino acid content.

Journal Article↗
PubMed1981

Subcellular Localization of 2-(beta-d-Glucosyloxy)-Cinnamic Acids and the Related beta-glucosidase in Leaves of Melilotus alba Desr.

The distribution of the glucosides of trans- and cis-2-hydroxy cinnamic acid and of the beta-glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts.The specific activities of the beta-glucosidase in the two tissues were also similar; thus, most of the enzyme is contained in mesophyll tissue. However, the amount of enzyme in mesophyll protoplast extracts amounts to only 1 to 2% of the activity present in leaf homogenates when chlorophyll was the basis for comparison. (This small amount of coumarin-beta-glucosidase present in protoplasts is not associated with chlorophyll-containing fractions.) In contrast, 90% of the uridine diphosphate glucose-o-coumaric acid glucosyl transferase activity present in leaf homogenate was recoverable in protoplasts prepared from intact leaves. Such results indicate that most of the coumarin-beta-glucosidase in M. alba leaves is located in the extracytoplasmic space. Only a small fraction (7%) of this extra cytoplasmic beta-glucosidase was associated with individual cells or cell clusters isolated from clover leaves.

Journal Article↗
PubMed1976

Phenolic metabolism in petunia tissues. IV. - Properties of p-coumarate : coenzyme A ligase isoenzymes.

Three p-coumarate: CoA ligases were separated from Petunia leaves. There was no interconversion from one form to another. The isoenzymes had a number of common properties: optimum pH, instability in the absence of polyols, action on p-coumaric acid as the common substrate. These enzymes differed significantly with respect to: --their substrate specificity towards the other C6-C3 units of Petunia. Form Ia (caffeate: CoA ligase) acted on caffeic acid, form Ib (sinapate: CoA ligase) on sinapic acid form II (ferulate: CoA ligase) on ferulic acid. --their thermal stability. --their sensitivity to phenolics: (a) caffeate: CoA ligase was inhibited by p-coumaroyl and caffeoyl quinic esters. It was insensitive to p-coumaroyl-glucose, on one hand and to a number of flavonoids on the other. (b) ferulate: CoA ligase was specifically inhibited by naringenin. (c) sinapate: CoA ligase was not inhibited by the selected compounds. In all cases, the inhibition was of the non competitive type and the enzymes were desensized to the modifier action by thermal treatment independently from the enzyme activity. These results suggest the occurrence of distinct sites of reception for the substrate and the inhibitor on the enzyme molecule. All these data are consistent with the hypothesis of the possible participation of each individual form in a limited number of pathways. This would be of physiological interest since the metabolic fate of the different cinnamic acids could be independently controlled at the p-coumarate: CoA ligase level.

Coenzyme A Ligases↗
PubMed1975

Phenolic metabolism in petunia tissues. I. Characteristic responses of enzymes involved in different steps of polyphenol synthesis to different hormonal influences.

Pronounced changes in enzymatic patterns occur in petunia tissues when calluses are subcultured on media containing different growth substances. As judged by variations of enzymes related to primary metabolism (6-phosphogluconate and malate dehydrogenases) there are individual responses for each metabolic pathway. Concerning the enzymes of aromatic metabolism: (a) Phenylalanine ammonia-lyase, cinnamate and p-coumarate hydroxylases and the enzyme(s) activating phenylpropanoid units vary in the same manner. (b) Chalcone-flavanone isomerase, a key enzyme in the synthesis of flavonoids, and coniferyl alcohol dehydrogenase, which leads to the monomers of lignins, have, on the other hand, an independent behaviour. These responses show that the enzymes involved in the synthesis and activation of phenylpropanoid units seem to act coordinately in plants. Moreover, the data suggest that the common pathway leading to the activated cinnamic acids and the specific metabolic steps of lignin and flavonoid synthesis are regulated in a different way.

Alcohol Oxidoreductases↗