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R Douce

Publications and source records attributed to R Douce.

At least 91 records · Page 5Linked to original sources
PubMed1994

The major biotinyl protein from Pisum sativum seeds covalently binds biotin at a novel site.

Seeds of Pisum sativum contain a biotinyl polypeptide called SBP65 that behaves as a putative sink for the free vitamin, representing more than 90% of the total protein-bound biotin in mature seeds. A cDNA encoding SBP65 was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing. Peptide sequencing also indicated binding of the biotin to lysine 103. The biotinylation domain of SBP65 differs markedly from that of presently known biotin enzymes. Molecular analysis of the protein sequence reveals an extremely hydrophilic protein containing several repeated motifs. These properties, as well as the temporal and spatial patterns of expression of this protein, suggest that SBP65 belongs to the LEA (late embryogenesis-abundant) group of proteins.

Amino Acid Sequence↗
PubMed1993

Glycine decarboxylase complex from higher plants. Molecular cloning, tissue distribution and mass spectrometry analyses of the T protein.

cDNA clones encoding the precursor of the T protein of the glycine decarboxylase complex have been isolated from a pea leaf cDNA library in lambda gt11. The longest cDNA insert of 1430 bp encodes a polypeptide of 408 amino acid residues of which 30 residues constitute an N-terminal cleavable presequence and 378 residues make up the mature protein. Several results confirmed the identity of the cDNA and the exactness of the predicted primary structure. Firstly, we purified the T protein to homogeneity and its mass was measured by mass spectrometry. The mass obtained (40966 +/- 5 Da) was the value predicted from the cDNA (40961 Da). Secondly, the purified T protein was chemically cleaved with cyanogen bromide and the peptide fragments were analysed by high-performance liquid chromatography/electrospray ionization mass spectrometry and/or fast-atom-bombardment mass spectrometry. The mass values of all the peptides generated by chemical cleavage and measured by these techniques were very close to the values calculated from the predicted primary structure. Thirdly, microsequencing of some of these peptides, which represent 35% of the total protein, fits perfectly with the primary structure deduced from the cDNA. In the present HPLC/electrospray ionization MS studies we never detected the presence of covalently bound tetrahydropteroylpolyglutamate (H4PteGlun), either in the native T protein or in the different peptide fragments generated by the chemical cleavage. The absence of H4PteGlun bound to the T protein in our experimental conditions demonstrates that H4PteGlun is not covalently linked to the T protein. Northern blot analysis showed that the steady-state level of the mRNA corresponding to the T protein was high in green leaves compared to the level in etiolated leaves (approximately 8-10-fold higher). Surprisingly, a non-negligible amount of mRNA corresponding to the T protein was present in roots whereas the mRNA encoding the H protein was not detectable. Western blot analysis showed that the P, L and T proteins of the glycine decarboxylase complex were present in roots whereas the H protein was not detectable. Southern hybridization to pea genomic DNA indicated the presence of a single gene encoding the T protein of the glycine decarboxylase complex in the haploid genome.

Amino Acid Sequence↗
PubMed1993

Biotin biosynthesis in higher plant cells. Identification of intermediates.

Biotin biosynthesis was investigated in lavender cell cultures (Lavandula vera L.). Two different biological assays and two different HPLC procedures were used to identify all the intermediates involved in biotin biosynthesis. The pathway for biotin biosynthesis could be analyzed starting with [3H]pimelic acid as precursor, leading to labelled biotin and even to labelled biotinylated enzymes. Intermediates known from the bacterial pathway (7-oxo-8-amino-pelargonic acid, 7,8-diamino-pelargonic acid, dethiobiotin) were present in detectable amounts. Pimelic acid activation to pimeloyl-CoA could be observed. In contrast to bacterial cells, an unknown stable labelled intermediate, named compound A, accumulated. This compound coeluted with an authentic sample of 9-mercaptodethiobiotin from HPLC with an anion-exchange column and was as effective as biotin in supporting the growth of the strain bioB105 of Escherichia coli. When 3H-labelled compound A was added to the growth medium of the lavender cells it was incorporated in an acidomycin-sensitive manner into biotin. [3H]Dethiobiotin was incorporated into both compound A and biotin. These results strongly suggest that, in higher plant cells, the reaction catalysed by biotin synthase may proceed in two distinct steps involving mercaptodethiobiotin (9-mercaptodethiobiotin?) as an intermediate.

Acyl Coenzyme A↗
PubMed1993

Branched-chain-amino-acid biosynthesis in plants: molecular cloning and characterization of the gene encoding acetohydroxy acid isomeroreductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress).

Towards the goal of gaining a better understanding of the molecular mechanisms controlling branched-chain-amino-acid biosynthesis in plants, we have isolated, sequenced and characterized a gene encoding acetohydroxy acid isomero-reductase (ketol-acid reductoisomerase) from Arabidopsis thaliana (thale cress). Comparison between the acetohydroxy acid isomeroreductase cDNA and the genomic sequence has allowed us to determine the exon structure of the coding region. The isolated acetohydroxy acid isomeroreductase gene is distributed over approx. 4.5 kbp and contains nine introns (79-347 bp). The transcriptional start site was found to be 52 bp upstream of the translational initiation site. Southern-blot analysis of A. thaliana genomic DNA shows that the acetohydroxy acid isomeroreductase is encoded by a single-copy gene.

Alcohol Oxidoreductases↗
PubMed1993

Lack of aconitase in glyoxysomes and peroxisomes.

The aim of this work was to find out whether aconitase [citrate (isocitrate) hydro-lyase, EC 4.2.1.3] which is rapidly inactivated by H2O2, is present in the microbodies from plant cells. The separation of intact organelles from castor-bean (Ricinus communis) endosperm and potato (Solanum tuberosum) tuber indicated that aconitase activity is essentially limited to the mitochondria and cytosol fraction, but was not detected in highly purified castor-bean endosperm and potato tuber peroxisomes. An isotropic e.p.r. signal of the type expected for the 3Fe cluster of oxidized aconitase was not detected in microbodies. In immunoblot analyses, antibodies raised against potato tuber mitochondrial aconitase did not cross-react with any glyoxysomal or peroxisomal protein. Positive reactions were found for cytosol fraction and mitochondria of castor-bean endosperm. The operation of the full glyoxylate cycle in isolated glyoxysomes requires the presence of aconitase in the incubation medium. It is concluded that glyoxysomes are probably devoid of aconitase and that the glyoxylate cycle requires a detour via the cytosol, which contains a powerful aconitase activity.

Aconitate Hydratase↗
PubMed1993

Common sequence motifs coding for higher-plant and prokaryotic O-acetylserine (thiol)-lyases: bacterial origin of a chloroplast transit peptide?

A comparison of the amino acid sequence of O-acetylserine (thiol)-lyase (EC 4.2.99.8) from Escherichia coli and the isoforms of this enzyme found in the cytosolic and chloroplastic compartments of spinach (Spinacia oleracea) leaf cells allows the essential lysine residue involved in the binding of the pyridoxal 5'-phosphate cofactor to be identified. The results of further sequence comparison of cDNAs coding for these proteins are discussed in the frame of the endosymbiotic theory of chloroplast evolution. The results are compatible with a mechanism in which the chloroplast enzyme originated from the cytosolic enzyme and both plant genes originated from a common prokaryotic ancestor. The comparison also suggests that the 5'-non-coding sequence of the bacterial gene was transferred to the plant cell nucleus and that it has been used to create the N-terminal portions of both plant enzymes, and possibly the transit peptide of the chloroplast enzyme.

Amino Acid Sequence↗
PubMed1993

Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and glycine decarboxylase from pea leaf mitochondria.

Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the glycine-cleavage system and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the glycine decarboxylase T-protein.

Amino Acid Oxidoreductases↗
PubMed1993

Localization of free and bound biotin in cells from green pea leaves.

Cytosol and vacuoles from protoplasts, chloroplasts, and mitochondria from green pea (Pisum sativum) leaves were purified and examined for their biotin content. The bulk of free biotin was shown to be exclusively associated with the cytosolic fraction at a concentration of about 4 pmol/mg protein and no bound biotin was detected. The bulk of bound biotin (biotin-containing carboxylases) was associated with the soluble fraction of chloroplasts and mitochondria at a concentration of about 1.2 and 13 microM, respectively. No free biotin was detected in these organelles. Western blot analysis of total, chloroplastic, and mitochondrial polypeptides, using horseradish peroxidase-labeled streptavidin, revealed three biotin-containing polypeptides with molecular mass of 220,000, 76,000 and 34,000. All were detected in the total pea leaf extract, but the M(r) 76,000 and the M(r) 34,000 biotinylated polypeptides were only detected in mitochondria and chloroplasts, respectively. 3-Methylcrotonyl-coenzyme A carboxylase and acetyl-coenzyme A carboxylase activities were measured in these two compartments, respectively. Previously, it has been shown that the M(r) 76,000 polypeptide was the biotinylated subunit of the mitochondrial 3-methylcrotonyl-coenzyme A carboxylase. In this paper, the origin and putative function of free biotin located in cytosol are discussed.

Biotin↗
PubMed1993

Transport of inorganic pyrophosphate across the spinach chloroplast envelope.

Spinach-leaf chloroplasts take up PPi at a rate of 1.9 mumol/h per mg of chlorophyll (Chl) in the dark and 1.6 mumol/h per mg of Chl in the light. The Km for PPi transport is 32 microM in the dark and 6 microM in the light. Uptake is inhibited by pyridoxal phosphate, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid and imidodiphosphate, but not by NaF or EDTA. PPi does not appear to cross the chloroplast envelope in exchange for Pi, suggesting that it is not transported by the phosphate translocator. Exchange of PPi and adenine nucleotides across the chloroplast envelope is very slow and PPi does not competitively inhibit ATP uptake, suggesting that little, if any, PPi is transported by the adenine-nucleotide translocator. These results are consistent with the presence of a specific, high-affinity PPi translocator in the spinach chloroplast envelope. It is proposed that in vivo PPi is taken up into the chloroplast from the cytosol to replenish the Pi pool in the stroma.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

13C nuclear magnetic resonance studies of malate and citrate synthesis and compartmentation in higher plant cells.

The synthesis of malate and citrate by sycamore cells (Acer pseudoplatanus L.) perfused with KH13CO3 was analyzed using 13C NMR. To perform in vivo experiments, cells were compressed in a 25-mm tube and perfused with an arrangement enabling tight control of the circulating nutrient medium. An original method using paramagnetic Mn2+ that induced a complete loss of the vacuolar malate and citrate signals was developed to discriminate between cytoplasmic and vacuolar pools of malate and citrate. Our results indicated the following. (a) The accumulation of appreciable amounts of malate in sycamore cells required rather high (1 mM) concentrations of bicarbonate at all the pH values tested. (b) Malate was equally labeled at C-1 and C-4, suggesting that malate labeled at C-1 was produced by randomization of C-1 and C-4 by mitochondrial fumarase. Indeed, the separation of the intact organelles from the lysed protoplasts indicated that fumarase activity was essentially limited to the mitochondria. Similarly, citrate was equally enriched at C-1 and C-5 + C-6 carboxyls. (c) Malate appeared first in the cytoplasmic compartment; and when a threshold of cytoplasmic malate concentration was attained, malate molecules were expelled into the vacuole, where they accumulated. On the other hand, citrate accumulated steadily in the vacuole. Pulse-chase experiments demonstrated the central role played by the tonoplast in governing the vacuolar influx of citrate and the permanent exchange of malate between the cytoplasm and the vacuole.

Bicarbonates↗
PubMed1993

Characterization of the primary structure of H-protein from Pisum sativum and location of a lipoic acid residue by combined liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

A purified extract of H-protein, a subunit of the glycine cleavage complex of the pea leaf mitochondria, was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using both continuous flow fast atom bombardment (CF-FAB) and electrospray ionization (ESI) mass spectrometry. Determination of the molecular weight of the entire protein, a 14 kDa subunit of the glycine decarboxylase complex, was achieved by ESI mass spectrometry and revealed covalent binding of the protein to the stabilizing agent beta-mercapto-ethanol. On-line LC/MS analysis of peptides arising from the endoproteinase Glu-C digestion of the H-protein was achieved using capillary columns (0.25 mm i.d.), and permitted confirmation of the previously reported sequence deduced from cDNA cloning experiments. The detailed interpretation of data extracted from these LC/MS experiments facilitated identification of peptides containing modified amino acid residues. In particular the identification of a lipoic acid cofactor, a rather unusual modified lysine residue which interacts with different active sites in the enzyme complex, was achieved using both LC/CF-FAB-MS and LC/ESI-MS. The exact location of this modified lysine residue was determined by obtaining fragment spectra of multiply protonated precursor ions of selected peptides, using on-line LC/MS/MS techniques.

Amino Acid Oxidoreductases↗
PubMed1993

Detection and characterization of a plastid envelope DNA-binding protein which may anchor plastid nucleoids.

Chloroplast DNA (cpDNA) binds to the envelope membrane of actively dividing chloroplasts (plastids) in young pea leaves. South-western blotting was used to identify and characterize the protein involved in the binding of cpDNA to the envelope membrane. A 130 kDa protein in the inner chloroplast (plastid) envelope membrane binds specific sequences within the cpDNA. These included a 0.41 kbp sequence located upstream of the psaAB gene, a 0.57 kbp sequence located downstream of the petA gene and a 1.2 kbp sequence located within the rpoC2 gene. The protein was detected in the envelope membrane of young pea leaves in which the cpDNA had been located by fluorescence microscopy at the chloroplast periphery, whereas it was undetectable in mature leaves. We therefore propose that the 130 kDa protein is involved in the binding of cpDNA to the envelope membrane, and named it plastid envelope DNA-binding protein.

Amino Acid Sequence↗
PubMed1993

O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform.

The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana.

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.

2-Acetolactate Mutase↗
PubMed1992

Isolation and kinetic properties of acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) chloroplasts overexpressed in Escherichia coli.

Acetohydroxy acid isomeroreductase catalyses a two-step reaction, an alkyl migration and a NADPH-dependent reduction, in the assembly of the carbon skeletons of branched-chain amino acids. Detailed investigations of acetohydroxy acid isomeroreductase aimed at elucidating the biosynthetic pathway of branched-chain amino acids and at designing new inhibitors of the enzyme having herbicidal potency have so far been conducted with the enzymes isolated from bacteria. To gain more information on a plant system, the gene encoding the mature acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) leaf chloroplasts has been used to transform Escherichia coli cells and to overexpress the enzyme. A rapid protocol is described that allows the preparation of large quantities of pure spinach chloroplast acetohydroxy acid isomeroreductase. Kinetic and structural properties of the plant enzyme expressed in Escherichia coli are compared with those reported in our previous studies on the native enzymes purified from spinach chloroplasts and with those reported for the corresponding enzymes isolated from Escherichia coli and Salmonella typhimurium. Both the plant and the bacterial enzymes obey an ordered mechanism in which NADPH binds first, followed by substrate (either 2-acetolactate or 2-aceto-2-hydroxybutyrate). Inhibition studies employing an inactive substrate analogue, 2-hydroxy-2-methyl-3-oxopentanoate, showed, however, that the binding of 2-hydroxy-2-methyl-3-oxopentanoate and NADPH occurs randomly, suggestive of some flexibility of the plant enzyme active site. The observed preference of the enzyme for 2-aceto-2-hydroxybutyrate over 2-acetolactate is discussed with regard to the contribution of acetohydroxy acid isomeroreductase activity in the partitioning between isoleucine and valine biosyntheses. Moreover, the kinetic properties of the chloroplast enzyme support the notion that biosynthesis of branched-chain amino acids in plants is controlled by light. As judged by analytical-ultracentrifugation and gel-filtration analyses the overexpressed plant enzyme is a dimer of identical subunits.

2-Acetolactate Mutase↗
PubMed1992

Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.

Cell Fractionation↗